Stable, aqueous formulations of antibodies that bind il5 receptor

ABSTRACT

The present invention relates to stable, aqueous antibody formulations. In some embodiments, the stable, aqueous formulations comprise about 2 mg/mL to about 100 mg/mL of an anti-IL5R antibody, and about 0.002% to about 0.01% polysorbate-20. Also provided are methods of making and methods of using such antibody formulations.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/298,474 filed Mar. 11, 2019 which is a continuation of U.S.application Ser. No. 14/521,870 filed Oct. 23, 2014 which claims benefitunder 35 U.S.C. § 119(e) of U.S. Provisional Application No. 61/895,143filed Oct. 24, 2013. The above listed applications are incorporated byreference herein in its entirety for all purposes.

REFERENCE TO THE SEQUENCE LISTING

This application incorporates by reference a Sequence Listing submittedwith this application as text file entitled IL5R-300-US-CNT2-Seq-Listcreated on Jun. 10, 2019 and having a size of 9.7 kb.

FIELD OF THE INVENTION

The present invention relates to stable, aqueous antibody formulations.In some embodiments, the stable, aqueous formulations comprise about 2mg/mL to about 100 mg/mL of an antibody, wherein the antibody comprisesa heavy chain variable region and a light chain variable region, whereinthe heavy chain variable region comprises the Kabat-defined CDR1, CDR2,and CDR3 sequences of SEQ ID NOs: 5-7, and wherein the light chainvariable region comprises the Kabat-defined CDR1, CDR2, and CDR3sequences of SEQ ID NOs: 8-10, and about 0.002% to about 0.01%polysorbate-20. Also provided are methods of making and methods of usingsuch antibody formulations.

BACKGROUND OF THE INVENTION

Antibodies have been used in the treatment of various diseases andconditions due to their specificity of target recognition, therebygenerating highly selective outcomes following systemic administration.In order for antibodies to remain effective, they must maintain theirbiological activity during their production, purification, transport andstorage. New production and purification techniques have been developedto provide for large amounts of highly purified monoclonal antibodies tobe produced. However, challenges still exist to stabilize theseantibodies for transport and storage, and yet even more challenges existto provide the antibodies in a dosage form suitable for administration.

Denaturation, aggregation, contamination, and particle formation can besignificant obstacles in the formulation and storage of antibodies. Dueto the wide variety of antibodies, there are no universal formulationsor conditions suitable for storage of all antibodies. Optimalformulations and conditions suitable for storage of one antibody areoften specific to that antibody. Thus, antibody storage formulations andmethods are often a significant part of the research and developmentprocess for a commercial antibody.

Various methods have been proposed to overcome the challenges associatedwith antibody stability. For example, in some instances, the antibody isoften lyophilized, and then reconstituted shortly before administration.However, reconstitution is generally not ideal, since it adds anadditional step to the administration process, and could introducecontaminants to the formulation. Additionally, even reconstitutedantibodies can suffer from aggregation and particle formation. Thus, aneed exists to provide stable, aqueous antibody formulations that canovercome the challenges associated with transport and storage.

BRIEF SUMMARY OF THE INVENTION

The present invention is directed to a stable, aqueous antibodyformulation comprising: (a) about 2 mg/mL to about 100 mg/mL of anantibody, wherein the antibody comprises a heavy chain variable regionand a light chain variable region, wherein the heavy chain variableregion comprises the Kabat-defined CDR1, CDR2, and CDR3 sequences of SEQID NOs: 5-7, and wherein the light chain variable region comprises theKabat-defined CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 8-10, and(b) about 0.002% to about 0.01% polysorbate-20.

In some embodiments, the antibody formulation further comprises anuncharged excipient. In some embodiments, the uncharged excipient istrehalose. In some embodiments, the uncharged excipient concentration isabout 20 mM to about 80 mM. In some embodiments, the uncharged excipientconcentration is about 200 mM to about 400 mM.

The antibody can be present in various concentrations. In someembodiments, the formulation comprises about 2 to about 20 mg/ml of theantibody. In some embodiments, the formulation comprises about 20 toabout 100 mg/ml of the antibody. In one embodiment, the formulationcomprises 30 mg/ml of the antibody.

The formulation can further comprise arginine. In some embodiments, thearginine is L-arginine. In some embodiments, the formulation comprisesabout 100 mM to about 200 mM L-arginine. In some embodiments, theformulation comprises about 120 mM to about 140 mM L-arginine, and about40 mM to about 60 mM uncharged excipient. In one embodiment, theformulation comprises about 125 mM L-arginine. In one embodiment, theformulation comprises about 130 mM L-arginine.

In some embodiments, the formulation further comprises histidine. Insome embodiments, the histidine concentration is about 15 mM to about 30mM. In one embodiment, the histidine concentration is about 20 mM.

In some embodiments, the antibody was not subjected to lyophilization.

In some embodiments, the invention is directed to a stable, aqueousantibody formulation comprising about 2 mg/mL to about 100 mg/mL of anantibody, wherein the antibody comprises a heavy chain variable regionand a light chain variable region, wherein the heavy chain variableregion comprises the Kabat-defined CDR1, CDR2, and CDR3 sequences of SEQID NOs: 5-7, and wherein the light chain variable region comprises theKabat-defined CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 8-10,wherein said formulation is stable upon storage at about 40° C. for atleast 1 month. In some embodiments, the formulation is stable uponstorage at about 25° C. for at least 3 months. In some embodiments, theformulation is stable upon storage at about 5° C. for at least 18months. In some embodiments, the antibody stored at about 40° C. for atleast 1 month retains at least 80% of binding ability to an IL-5Rpolypeptide compared to a reference antibody which has not been stored.In some embodiments, the antibody stored at about 5° C. for at least 6months retains at least 80% of binding ability to an IL-5R polypeptidecompared to a reference antibody which has not been stored. In someembodiments, the antibody stored at about 40° C. for at least 1 monthretains at least 95% of binding ability to an IL-5R polypeptide comparedto a reference antibody which has not been stored. In some embodiments,the antibody stored at about 5° C. for at least 6 months retains atleast 95% of binding ability to an IL-5R polypeptide compared to areference antibody which has not been stored. In some embodiments, lessthan 2% of the antibody forms an aggregate upon storage at about 40° C.for at least 1 month as determined by as determined by HPSEC. In someembodiments, less than 2% of the antibody forms an aggregate uponstorage at about 5° for at least 12 months as determined by HPSEC.

In some embodiments, the formulation is substantially free fromparticles upon storage at about 40° C. for at least 1 month asdetermined by visual inspection. In some embodiments, the formulation issubstantially free from particles upon storage at about 5° C. for atleast 12 months as determined by visual inspection.

In some embodiments, the formulation is an injectable formulation. Insome embodiments, the formulation is suitable for intravenous,subcutaneous, or intramuscular administration.

In some embodiments, the invention is directed to a sealed containercontaining an antibody formulation as described herein. In someembodiments, the invention is directed to a pharmaceutical unit dosageform suitable for parenteral administration to a human which comprisesan antibody formulation as described herein in a suitable container. Insome embodiments, the antibody formulation is administeredintravenously, subcutaneously, or intramuscularly. In some embodiments,the suitable container is a pre-filled syringe. In some embodiments, thepre-filled syringe comprises a needle. In some embodiments, the needleis a 29 G thin wall needle. In some embodiments, the pre-filled syringeis a plastic syringe or a glass syringe. In some embodiments, thepre-filled syringe is made of materials that are substantially free fromtungsten.

In some embodiments, the pre-filled syringe is coated with silicone. Insome embodiments, the pre-filled syringe comprises a plunger having afluoropolymer resin disk. In some embodiments, the pre-filled syringecomprises (a) about 2 mg/mL to about 20 mg/mL of an antibody, whereinthe antibody comprises a heavy chain variable region and a light chainvariable region, wherein the heavy chain variable region comprises theKabat-defined CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 5-7, andwherein the light chain variable region comprises the Kabat-definedCDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 8-10, and (b) about 0.002%to about 0.01% polysorbate-20. In some embodiments, the pre-filledsyringe further comprises: (c) about 40 mM to about 60 mM trehalose, and(d) about 110 mM to about 150 mM L-arginine. In some embodiments, theinvention is directed to a pre-filled syringe comprising: (a) about 20mg/mL to about 100 mg/mL of an antibody, wherein the antibody comprisesa heavy chain variable region and a light chain variable region, whereinthe heavy chain variable region comprises the Kabat-defined CDR1, CDR2,and CDR3 sequences of SEQ ID NOs: 5-7, and wherein the light chainvariable region comprises the Kabat-defined CDR1, CDR2, and CDR3sequences of SEQ ID NOs: 8-10, and (b) about 0.002% to about 0.01%polysorbate-20. In some embodiments, the formulation further comprises:(c) about 200 mM to about 300 mM trehalose.

In some embodiments, the invention is directed to a kit comprising theformulation described herein, the container described herein, the unitdosage forms described herein, or the pre-filled syringes describedherein.

In some embodiments, the invention is directed to a stable, aqueousantibody formulation comprising: (a) about 2 mg/mL to about 20 mg/mL ofan antibody, wherein the antibody comprises a heavy chain variableregion and a light chain variable region, wherein the heavy chainvariable region comprises the Kabat-defined CDR1, CDR2, and CDR3sequences of SEQ ID NOs: 5-7, and wherein the light chain variableregion comprises the Kabat-defined CDR1, CDR2, and CDR3 sequences of SEQID NOs: 8-10, (b) about 0.002% to about 0.01% polysorbate-20, (c) about40 mM to about 60 mM trehalose, (d) about 110 mM to about 150 mML-arginine, and (e) about 15 to about 30 mM histidine. In oneembodiment, the invention is directed to a stable, aqueous antibodyformulation comprising: (a) about 2 mg/mL to about 20 mg/mL of anantibody, wherein the antibody comprises a heavy chain variable regionand a light chain variable region, wherein the heavy chain variableregion comprises the Kabat-defined CDR1, CDR2, and CDR3 sequences of SEQID NOs: 5-7, and wherein the light chain variable region comprises theKabat-defined CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 8-10, (b)about 0.006% polysorbate-20, (c) about 50 mM trehalose, (d) about 130 mML-arginine, and (e) about 20 mM histidine.

In some embodiments, the invention is directed to a stable, aqueousantibody formulation comprising: (a) about 20 mg/mL to about 100 mg/mLof an antibody, wherein the antibody comprises a heavy chain variableregion and a light chain variable region, wherein the heavy chainvariable region comprises the Kabat-defined CDR1, CDR2, and CDR3sequences of SEQ ID NOs: 5-7, and wherein the light chain variableregion comprises the Kabat-defined CDR1, CDR2, and CDR3 sequences of SEQID NOs: 8-10, (b) about 0.002% to about 0.01% polysorbate-20, (c) about200 mM to about 300 mM trehalose, and (d) about 15 to about 30 mMhistidine. In one embodiment, the invention is directed to a stable,aqueous antibody formulation comprising: (a) about 20 mg/mL to about 100mg/mL of an antibody, wherein the antibody comprises a heavy chainvariable region and a light chain variable region, wherein the heavychain variable region comprises the Kabat-defined CDR1, CDR2, and CDR3sequences of SEQ ID NOs: 5-7, and wherein the light chain variableregion comprises the Kabat-defined CDR1, CDR2, and CDR3 sequences of SEQID NOs: 8-10, (b) about 0.006% polysorbate-20, (c) about 250 mMtrehalose, and (d) about 20 mM histidine. In another embodiment, theinvention is directed to a stable, aqueous antibody formulationcomprising: (a) about 30 mg/mL of an antibody, wherein the antibodycomprises a heavy chain variable region and a light chain variableregion, wherein the heavy chain variable region comprises theKabat-defined CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 5-7, andwherein the light chain variable region comprises the Kabat-definedCDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 8-10, (b) about 0.006%polysorbate-20, (c) about 250 mM trehalose, and (d) about 20 mMhistidine.

In some embodiments, the invention is directed to a method of producinga stable, aqueous antibody formulation, the method comprising: (a)purifying an antibody to about 1 mg/mL to about 400 mg/mL, wherein theantibody comprises a heavy chain variable region and a light chainvariable region, wherein the heavy chain variable region comprises theKabat-defined CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 5-7, andwherein the light chain variable region comprises the Kabat-definedCDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 8-10; (b) placing theisolated antibody in a stabilizing formulation to form the stable,aqueous antibody formulation, wherein the resulting stable, aqueousantibody formulation comprises: (i) about 2 mg/mL to about 100 mg/mL ofthe antibody, and (ii) about 0.002% to about 0.01% polysorbate-20.

In some embodiments, the invention is directed to a method of making astable, aqueous antibody formulation, the method comprising: (a)purifying an antibody to about 1 mg/mL to about 400 mg/mL, wherein theantibody comprises a heavy chain variable region and a light chainvariable region, wherein the heavy chain variable region comprises theKabat-defined CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 5-7, andwherein the light chain variable region comprises the Kabat-definedCDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 8-10; (b) diluting theantibody to about 2 mg/mL to about 20 mg/mL of the antibody into asolution comprising: (i) about 0.002% to about 0.01% polysorbate-20,(ii) about 40 mM to about 60 mM trehalose, and (iii) about 110 mM toabout 150 mM L-arginine.

In some embodiments, the invention is directed to a method of making astable, aqueous antibody formulation, the method comprising: (a)purifying an antibody to about 1 mg/mL to about 400 mg/mL, wherein theantibody comprises a heavy chain variable region and a light chainvariable region, wherein the heavy chain variable region comprises theKabat-defined CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 5-7, andwherein the light chain variable region comprises the Kabat-definedCDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 8-10; (b) diluting theantibody to about 20 mg/mL to about 100 mg/mL of the antibody into asolution comprising: (i) about 0.002% to about 0.01% polysorbate-20, and(ii) about 200 mM to about 300 mM trehalose.

In some embodiments, the invention is directed to a method of producinga reconstituted antibody formulation comprising an antibody, wherein theantibody comprises a heavy chain variable region and a light chainvariable region, wherein the heavy chain variable region comprises theKabat-defined CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 5-7, andwherein the light chain variable region comprises the Kabat-definedCDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 8-10, the methodcomprising: (a) purifying the antibody from a cell culture; (b)lyophilizing the isolated antibody; (c) adding the lyophilized antibodyto a aqueous solution to form a reconstituted antibody formulation,wherein the reconstituted antibody formulation comprises: (i) about 2mg/mL to about 100 mg/mL of the antibody, and (ii) about 0.002% to about0.01% polysorbate-20.

In some embodiments, the invention is directed to an antibodyformulation comprising an antibody, wherein the antibody comprises aheavy chain variable region and a light chain variable region, whereinthe heavy chain variable region comprises the Kabat-defined CDR1, CDR2,and CDR3 sequences of SEQ ID NOs: 5-7, and wherein the light chainvariable region comprises the Kabat-defined CDR1, CDR2, and CDR3sequences of SEQ ID NOs: 8-10, wherein the antibody formulation isessentially free of particles. In some embodiments, the antibodyformulation comprises an antibody, wherein the antibody comprises aheavy chain variable region and a light chain variable region, whereinthe heavy chain variable region comprises the Kabat-defined CDR1, CDR2,and CDR3 sequences of SEQ ID NOs: 5-7, and wherein the light chainvariable region comprises the Kabat-defined CDR1, CDR2, and CDR3sequences of SEQ ID NOs: 8-10, wherein the antibody formulation isessentially free of active glutathione S-transferase (GST). In someembodiments, the antibody formulation is essentially free of GST. Insome embodiments, the antibody formulation is essentially free ofparticles for at least 1 month when stored at 38-42° C. In someembodiments, the antibody formulation is essentially free of particlesfor at least 6 months when stored at 2-6° C. In some embodiments, theantibody formulation is essentially free of particles for at least 18months when stored at 2-6° C.

In some embodiments, the invention is directed to a method of purifyingan antibody, wherein the antibody comprises a heavy chain variableregion and a light chain variable region, wherein the heavy chainvariable region comprises the Kabat-defined CDR1, CDR2, and CDR3sequences of SEQ ID NOs: 5-7, and wherein the light chain variableregion comprises the Kabat-defined CDR1, CDR2, and CDR3 sequences of SEQID NOs: 8-10, the method comprising: (a) obtaining a cell culturecomprising the antibody, (b) performing affinity chromatography on theantibody (c) performing cation exchange on the antibody, (d) performingmixed-mode chromatography on the antibody. In some embodiments, themethod further comprises a viral inactivation process and/or adiafiltration process.

In some embodiments, the invention is directed to a method of treating apulmonary disease or disorder in a subject, the method comprisingadministering a therapeutically effective amount of the antibodyformulations described herein, the containers described herein, the unitdosage forms described herein, or the pre-filled syringes describedherein. In some embodiments, the pulmonary disease or disorder is aneosinophilic disease or disorder. In some embodiments, the pulmonarydisease or disorder is asthma, COPD, eosinophilic asthma, combinedeosinophilic and neutrophilic asthma, aspirin sensitive asthma, allergicbronchopulmonary aspergillosis, acute and chronic eosinophilicbronchitis, acute and chronic eosinophilic pneumonia, Churg-Strausssyndrome, hypereosinophilic syndrome, drug, irritant andradiation-induced pulmonary eosinophilia, infection-induced pulmonaryeosinophilia (fungi, tuberculosis, parasites), autoimmune-relatedpulmonary eosinophilia, eosinophilic esophagitis, Crohn's disease, orcombination thereof. In some embodiments, the pulmonary disease ordisorder is asthma. In some embodiments, the pulmonary disease ordisorder is chronic obstructive pulmonary disease (COPD).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Amino acid sequences of anti-IL5R antibody.

FIG. 2 represents the effect of polysorbate-20 on fraction of monomer insolution. A polysorbate concentration above 0.005% is required tocompletely maintain the monomer level.

FIG. 3 represents the effect of polysorbate-20 on sub-visible particlecounts in 2 g/L solutions. Data for particles>2 μm is not shown butexhibits a similar pattern to the larger particles. The data indicatesthat sub-visible particle levels in 2 g/L solutions are not controlledby any level of polysorbate.

FIG. 4 represents the effect of polysorbate-20 on sub-visible particlecounts in 100 g/L solutions. Data for particles>2 μm is not shown butexhibits a similar pattern to the larger particles. The data indicatesthat polysorbate levels above 0.003% control sub-visible particle levelsin 100 g/L solutions.

FIG. 5. HPLC monomer (%) and other (%) results as a function of solutionpH and protein concentration. Monomer loss is minimized in the pH rangeof 5.5-6.5.

FIG. 6. Particle formation including sub-visible particles>10 μmmeasured by MFI and visible particles evaluated by comparison tostandards as a function of solution pH and protein concentration.Subvisible particles counts are dependent on protein concentration butshow no clear trend with pH. More visible particles are observed in thelower protein concentration solutions in the pH range from 5.5-6.5.

FIG. 7. Sub-visible particle counts>10 μm, aspect ratio filtered toremove silicone oil droplets. The data compares SVP counts just aftershipping to those after 1 month of storage at 25° C. High counts areseen for low protein concentrations, independent of PS-20.

FIG. 8. Particle counts>10 μm by MFI after simulated transportation forvarying protein concentrations and formulations. Higher ionic strengthformulations are more stable, as are trehalose formulations>10 g/L.

FIG. 9. Particle counts>10 μm by MFI after simulated transportation for2 g/L protein and varying formulations. The arginine concentrationshould be >50 mM and the NaCl concentration should be >75 mM.

FIG. 10. Particle counts>10 μm by MFI after simulated transportation for2 g/L protein and varying formulations. Any excipient concentration inthis range is acceptable.

FIG. 11. Particle counts>10 μm by MFI after simulated transportation for2 g/L protein and varying formulations.

FIG. 12(A-C). Monomer loss for the 2 mg/mL, 20 mg/mL and 100 mg/mLformulations in vials and pre-filled syringes are shown. All of the PFSshow similar loss to the vials and to one another.

FIG. 13. A graphical representation of the bracketing strategy is shown.Blue indicates the arginine or NaCl containing formulations. Greenindicates the trehalose formulation. Data points are prepared samplesand lines indicate the bracketed options available to achieveintermediate doses.

FIG. 14. The testing plan for stability study #2. All the marked testswill be run on the antibody formulation. Yellow shading indicates thetest will be run on the placebo. The notation “ABC” indicates submissionfor the following tests: potency (BIOASSAY), RP-HPLC, cIEF, non-reducedBioAnalyzer, and reduced BioAnalyzer.

FIG. 15 is a graph showing samples prepared to define the design spaceas a function of protein and polysorbate-20 (“PS-20”) concentrations forboth formulation brackets.

FIG. 16 is a graph of sub-visible particles by MFI at time 0, aftershipping. Results show that particles form if no PS-20 is present, but0.002% PS-20 is sufficient to inhibit particle formation upon shipping.

FIG. 17(A-D). Visible particle observations are scored againstappearance standards and shown here at the 9 month time point. Theobservations were made very close to the light. Samples shown includepre-filled syringes (“PFS”) containing the trehalose formulation (FIG.3A), vials containing the trehalose formulation (FIG. 3B), PFScontaining the arginine formulation (FIG. 3C), and vials containing thearginine formulation (FIG. 3D). The data supports a target of 0.006%PS-20 and an acceptable range of 0.002-0.01% PS-20 in PFS. Vials areshown as a worst case comparison.

FIG. 18 compares various SVP methods for capturing an increase withsample time, correlating with visible particles. Flow cytometry andsmall particle counts (>1 and >2 μm) by MFI are able to capture thetrend well.

FIG. 19(A-B) is a comparison of Appearance standard scores and MFIresults (particles>1 μm) for trehalose formulations in PFS (FIG. 5A) andvials (FIG. 5B). Good agreement is observed for the two methods, whichboth indicate the acceptable range of PS-20 is 0.002-0.01%.

FIG. 20 represents a bracket design as outlined in Example 3 and thelead lot stability study performed in ABC. Orange shaded area indicatesthe arginine formulation and blue shaded area indicates the trehaloseformulation, all with 0.006% PS-20.

FIG. 21 represents one schematic example of an antibody purificationprocess.

FIG. 22 represents 2D gel analysis of the flow-thru of a Protein-Acolumn used in the purification of anti-IL5R antibody.

DETAILED DESCRIPTION OF THE INVENTION

It should be appreciated that the particular implementations shown anddescribed herein are examples, and are not intended to otherwise limitthe scope of the application in any way. It should also be appreciatedthat each of the embodiments and features of the invention describedherein can be combined in any and all ways.

The published patents, patent applications, websites, company names, andscientific literature referred to herein are hereby incorporated byreference in their entirety to the same extent as if each wasspecifically and individually indicated to be incorporated by reference.Any conflict between any references cited herein and the specificteachings of this specification shall be resolved in favor of thelatter. Likewise, any conflict between an art-understood definition of aword or phrase and a definition of the word or phrase as specificallytaught in this specification shall be resolved in favor of the latter.

As used in this specification, the singular forms “a,” “an” and “the”specifically also encompass the plural forms of the terms to which theyrefer, unless the content clearly dictates otherwise.

Throughout the present disclosure, all expressions of percentage, ratio,and the like are “by weight” unless otherwise indicated. As used herein,“by weight” is synonymous with the term “by mass,” and indicates that aratio or percentage defined herein is done according to weight ratherthan volume, thickness, or some other measure.

The term “about” is used herein to mean approximately, in the region of,roughly, or around. When the term “about” is used in conjunction with anumerical range, it modifies that range by extending the boundariesabove and below the numerical values set forth. In general, the term“about” is used herein to modify a numerical value above and below thestated value by a variance of 10%.

Technical and scientific terms used herein have the meaning commonlyunderstood by one of skilled in the art to which the present applicationpertains, unless otherwise defined. Reference is made herein to variousmethodologies and materials known to those of skilled in the art.Standard reference works setting forth the general principles ofrecombinant DNA technology include Sambrook et al., “Molecular Cloning:A Laboratory Manual,” 2nd Ed., Cold Spring Harbor Laboratory Press, NewYork (1989); Kaufman et al., Eds., “Handbook of Molecular and CellularMethods in Biology in Medicine,” CRC Press, Boca Raton (1995); andMcPherson, Ed., “Directed Mutagenesis: A Practical Approach,” IRL Press,Oxford (1991), the disclosures of each of which are incorporated byreference herein in their entireties.

The present invention is directed to stable, aqueous antibodyformulations. As described herein, the term “antibody formulation”refers to a composition comprising one or more antibody molecules. Theterm “antibody” in the present invention is not particularly limited.For clarity, an “antibody” is taken in its broadest sense and includesany immunoglobulin (Ig), active or desired variants thereof, and activeor desirable fragments thereof (e.g., Fab fragments, camelid antibodies(single chain antibodies), and nanobodies). The term “antibody” can alsorefer to dimers or multimers. The antibody can be polyclonal ormonoclonal and can be naturally-occurring or recombinantly-produced.Thus, human, non-human, humanized, and chimeric antibodies are allincluded with the term “antibody.” Typically the antibody is amonoclonal antibody of one of the following classes: IgG, IgE, IgM, IgD,and IgA; and more typically is an IgG or IgA.

An antibody of the invention can be from any animal origin includingbirds and mammals. In some embodiments, the antibody of the methods ofthe invention are human, murine (e.g., mouse and rat), donkey, sheep,rabbit, goat, guinea pig, camel, horse, or chicken. As used herein,“human” antibodies include antibodies having the amino acid sequence ofa human immunoglobulin and include antibodies isolated from humanimmunoglobulin libraries or from animals transgenic for one or morehuman immunoglobulin and that do not express endogenous immunoglobulins.See, e.g., U.S. Pat. No. 5,939,598 by Kucherlapati et al.

An antibody of the invention can include, e.g., native antibodies,intact monoclonal antibodies, polyclonal antibodies, multispecificantibodies (e.g., bispecific antibodies) formed from at least two intactantibodies, antibody fragments (e.g., antibody fragments that bind toand/or recognize one or more antigens), humanized antibodies, humanantibodies (Jakobovits et al., Proc. Natl. Acad. Sci. USA 90:2551(1993); Jakobovits et al., Nature 362:255-258 (1993); Bruggermann etal., Year in Immunol. 7:33 (1993); U.S. Pat. Nos. 5,591,669 and5,545,807), antibodies and antibody fragments isolated from antibodyphage libraries (McCafferty et al., Nature 348:552-554 (1990); Clacksonet al., Nature 352:624-628 (1991); Marks et al., J. Mol. Biol.222:581-597 (1991); Marks et al., Bio/Technology 10:779-783 (1992);Waterhouse et al., Nucl. Acids Res. 21:2265-2266 (1993)). An antibodypurified by the method of the invention can be recombinantly fused to aheterologous polypeptide at the N- or C-terminus or chemicallyconjugated (including covalently and non-covalently conjugations) topolypeptides or other compositions. For example, an antibody purified bythe method of the present invention can be recombinantly fused orconjugated to molecules useful as labels in detection assays andeffector molecules such as heterologous polypeptides, drugs, or toxins.See, e.g., PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S.Pat. No. 5,314,995; and EP 396,387.

In some embodiments, the antibody formulation of the present inventioncomprises an anti-IL5 receptor (anti-IL5R) antibody. Antibodies of thepresent invention specifically bind to an antigen of interest or afragment thereof, and do not specifically bind to other antigens orfragments thereof. For example, an anti-IL5R antibody willimmunospecifically bind to an interleukin-5 receptor polypeptide anddoes not specifically bind to other polypeptides. Preferably, antibodiesor antibody fragments that immunospecifically bind to an IL-5 receptorhave a higher affinity to an IL-5 receptor or a fragment of an IL-5receptor polypeptide when compared to the affinity to other polypeptidesor fragments of other polypeptides. The affinity of an antibody is ameasure of its binding with a specific antigen at a singleantigen-antibody site, and is in essence the summation of all theattractive and repulsive forces present in the interaction between theantigen-binding site of an antibody and a particular epitope. Theaffinity of an antibody to a particular antigen (e.g., an IL-5polypeptide or fragment of an IL-5 polypeptide) may be expressed by theequilibrium constant K, defined by the equation K=[Ag Ab]/[Ag][Ab],which is the affinity of the antibody-combining site where [Ag] is theconcentration of free antigen, [Ab] is the concentration of freeantibody and [Ag Ab] is the concentration of the antigen-antibodycomplex. Where the antigen and antibody react strongly together therewill be very little free antigen or free antibody, and hence theequilibrium constant or affinity of the antibody will be high. Highaffinity antibodies are found where there is a good fit between theantigen and the antibody (for a discussion regarding antibody affinity,see Sigal and Ron ed., 1994, Immunology and Inflammation—BasicMechanisms and Clinical Consequences, McGraw-Hill, Inc. New York atpages 56-57; and Seymour et ah, 1995, Immunology—An Introduction for theHealth Sciences, McGraw-Hill Book Company, Australia at pages 31-32).Preferably, antibodies or antibody fragments that immunospecificallybind to an IL-5 polypeptide or fragment thereof do not cross-react withother antigens. That is, antibodies or antibody fragments thatimmunospecifically bind to an IL-5 polypeptide or fragment thereof witha higher energy than to other polypeptides or fragments of otherpolypeptides (see, e.g., Paul ed., 1989, Fundamental Immunology, 2^(nd)ed., Raven Press, New York at pages 332-336 for a discussion regardingantibody specificity). Antibodies or antibody fragments thatimmunospecifically bind to an IL-5 polypeptide can be identified, forexample, by immunoassays such as radioimmunoassays (RIAs), enzyme-linkedimmunosorbent assays (ELISAs), and BIAcore assays or other techniquesknown to those of skill in the art (see, e.g., Seymour et al, 1995,Immunology—An Introduction for the Health Sciences, McGraw-Hill BookCompany, Australia at pages 33-41 for a discussion of various assays todetermine antibody-antigen interactions in vivo). Antibodies or antibodyfragments that immunospecifically bind to an IL-5 polypeptide orfragment thereof only antagonize an IL-5 polypeptide and do notsignificantly antagonize other activities.

In one embodiment, an IL-5R polypeptide is human IL-5R, an analog,derivative or a fragment thereof. The nucleotide sequence of human IL-5Rcan be found in the GenBank database (see, e.g., Accession No.M96652.1). The amino acid sequence of human IL-5R can be found in theGenBank database (see, e.g., Accession No. Q01344). Each of theseAssession numbers is expressly incorporated by reference herein.

In some embodiments, the antibody formulation comprises an anti-IL5Rantibody, for example, a human anti-IL5R antibody. In some embodiments,the anti-IL5R antibody comprises a light chain comprising SEQ ID NO:2and a heavy chain comprising SEQ ID NO:4. In some further embodiments,the anti-IL5R antibody comprises a light chain variable regioncomprising SEQ ID NO:1 and a heavy chain variable region comprising SEQID NO:3. In a further embodiment, the anti-IL5R antibody comprises aheavy chain variable region and a light chain variable region, whereinthe heavy chain variable region comprises the Kabat-defined CDR1, CDR2,and CDR3 sequences of SEQ ID NOs: 5-7, and wherein the light chainvariable region comprises the Kabat-defined CDR1, CDR2, and CDR3sequences of SEQ ID NOs: 8-10. Those of ordinary skill in the art wouldeasily be able to identify Chothia-defined, Abm-defined or other CDRs.

In one embodiment, the anti-IL5R antibody is benralizumab. Informationregarding benralizumab (or fragments thereof) for use in the methodsprovided herein can be found in U.S. Patent Application Publication No.US 2010/0291073 A1, the disclosure of which is incorporated herein byreference in its entirety.

As used herein, the term “analog” or “antibody analog” in the context ofan antibody refers to a second antibody, i.e., antibody analog, thatpossesses a similar or identical functions as the antibody, but does notnecessarily comprise a similar or identical amino acid sequence of theantibody, or possess a similar or identical structure of the antibody.An antibody that has a similar amino acid sequence refers to an antibodyanalog that satisfies at least one of the following: (a) an antibodyanalog having an amino acid sequence that is at least 30%, at least 35%,at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95% or at least 99% identical to the amino acidsequence of the antibody; (b) an antibody analog encoded by a nucleotidesequence that hybridizes under stringent conditions to a nucleotidesequence encoding the antibody of at least 5 contiguous amino acidresidues, at least 10 contiguous amino acid residues, at least 15contiguous amino acid residues, at least 20 contiguous amino acidresidues, at least 25 contiguous amino acid residues, at least 40contiguous amino acid residues, at least 50 contiguous amino acidresidues, at least 60 contiguous amino residues, at least 70 contiguousamino acid residues, at least 80 contiguous amino acid residues, atleast 90 contiguous amino acid residues, at least 100 contiguous aminoacid residues, at least 125 contiguous amino acid residues, or at least150 contiguous amino acid residues; and (c) an antibody analog encodedby a nucleotide sequence that is at least 30%, at least 35%, at least40%, at least 45%, at least 50%, at least 55%, at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95% or at least 99% identical to the nucleotide sequenceencoding the antibody. An antibody analog with similar structure to theantibody refers to a proteinaceous agent that has a similar secondary,tertiary or quaternary structure to the antibody. The structure of anantibody analog or antibody can be determined by methods known to thoseskilled in the art, including but not limited to, peptide sequencing,X-ray crystallography, nuclear magnetic resonance, circular dichroism,and crystallographic electron microscopy.

To determine the percent identity of two amino acid sequences or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in the sequence of a first aminoacid or nucleic acid sequence for optimal alignment with a second aminoacid or nucleic acid sequence). The amino acid residues or nucleotidesat corresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences (i.e., % identity=numberof identical overlapping positions/total number of positions×100%). Inone embodiment, the two sequences are the same length.

The determination of percent identity between two sequences can also beaccomplished using a mathematical algorithm. One, non-limiting exampleof a mathematical algorithm utilized for the comparison of two sequencesis the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci.U.S.A. 87:2264-2268, modified as in Karlin and Altschul, 1993, Proc.Natl. Acad. Sci. U.S.A. 90:5873-5877. Such an algorithm is incorporatedinto the NBLAST and XBLAST programs of Altschul et ah, 1990, J. Mol.Biol. 215:403. BLAST nucleotide searches can be performed with theNBLAST nucleotide program parameters set, e.g., for score=100,wordlength=12 to obtain nucleotide sequences homologous to a nucleicacid molecules of the present invention. BLAST protein searches can beperformed with the XBLAST program parameters set, e.g., to score-50,wordlength=3 to obtain amino acid sequences homologous to a proteinmolecule of the present invention. To obtain gapped alignments forcomparison purposes, Gapped BLAST can be utilized as described inAltschul et al, 1997, Nucleic Acids Res. 25:3389-3402. Alternatively,PSI-BLAST can be used to perform an iterated search which detectsdistant relationships between molecules (Id). When utilizing BLAST,Gapped BLAST, and PSI-Blast programs, the default parameters of therespective programs (e.g., of XBLAST and NBLAST) can be used (see, e.g.,the NCBI website). Another preferred, non-limiting example of amathematical algorithm utilized for the comparison of sequences is thealgorithm of Myers and Miller, 1988, CABIOS 4:11-17. Such an algorithmis incorporated in the ALIGN program (version 2.0) which is part of theGCG sequence alignment software package. When utilizing the ALIGNprogram for comparing amino acid sequences, a PAM 120 weight residuetable, a gap length penalty of 12, and a gap penalty of 4 can be used.

In some embodiments, the antibody in the antibody formulation ispurified prior to being added to the antibody formulation. The terms“isolate,” and “purify” refer to separating the antibody from animpurity or other contaminants in the composition which the antibodyresides, e.g., a composition comprising host cell proteins. In someembodiments, at least 50%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5%, or 99.9%(w/w) of an impurity is purified from the antibody. For example, in someembodiments, purification of an antibody, e.g. anti-IL5R antibody, wouldcomprise separating the antibody from 99% (w/w) of the host cellproteins present originally in the composition.

In some embodiments, the terms “isolate,” and “purify” refer toseparating an antibody, e.g. anti-IL5R antibody, from an impurity orother contaminants in the composition to an extent consistent withguidelines of a governmental organization, e.g., the World HealthOrganization or the United States Food and Drug Administration.

Methods of purifying an antibody are known to those of skill in the art.Suitable techniques for carrying out purification include various typesof chromatography, such as affinity chromatography, hydrophobicinteraction, ion exchange (such as cation exchange chromatography ormixed-mode chromatography), and filtration.

Affinity chromatography refers to a separation method whereby anantibody, by virtue of its specific binding properties, is bound to anaffinity ligand for the antibody. The functional affinity ligand can beimmobilized on a solid or semi-solid support so that when a compositioncomprising the antibody is passed over the ligand and the solid support,the antibody having a specific binding affinity to the ligand adsorbs tothe ligand, and one or more other impurities are not adsorbed (or arebound at a lower affinity) and are separated from the antibody. Examplesof impurities that do not typically bind (or do not bind well) includeprocess-related impurities (e.g., host cell proteins, DNA, mediumcomponents) and some product-related impurities (e.g., antibodyfragments). In some embodiments, the solid support comprising the ligandis washed one or more times with a buffer to remove additionalimpurities before the adsorbed antibody is removed from the ligand andthe support. After one or more impurities have been removed, theadsorbed antibody can be removed (eluted) from the ligand and thesupport, resulting in isolation of the antibody from the originalcomposition. Methods of removing the antibody from the ligand andsupport are dependent on the ligand and are known to those of skill inthe art and can include, e.g., changes in environment, e.g., pH,addition or chaotropic agents or denaturants, or addition ofcommercially available elution buffers. In some embodiments, more thanone affinity purification process can be employed on an antibodycomposition. Various affinity ligands are known in the art, includingProtein A and Protein G (and combinations thereof). Immobilized ligandsare commercially available. For example, Protein A affinity systemsinclude MabSelect, MabSelect SuRe, MabSelect Xtra, MabSelect SuRe LX,Sepaharose CL-4B, ProSep vA, ProSep vA Ultra, and Ceramic HyperD.

Ion exchange chromatography includes cation exchange chromatography andmixed chromatography. Cation exchange chromatography refers to anymethod by which an antibody and some impurity or impurities can beseparated based on charge differences using a cation exchange matrix. Acation exchange matrix generally comprises covalently bound, negativelycharged groups. Weak or strong cation exchange resins may be employed.Commonly, strong cation exchange resins comprise supported organicgroups comprising sulphonic acid or sulphonate groups, depending uponthe pH. Weak cation exchanger resins commonly comprise supported organicgroups comprising carboxylic acid or carboxylate groups, depending uponthe pH. In certain embodiments, multimodal cation exchange resins can beused, which incorporate additional binding mechanisms as well as theionic interactions, for example one or more of hydrogen bondinginteractions and hydrophobic interactions. Examples of suitable cationexchange resins are well known in the art, and can include, but are notlimited to Fractogel, carboxymethyl (CM), sulfoethyl(SE),sulfopropyl(SP), phosphate (P) and sulfonate(S), PROPAC WCX-10™(Dionex), Capto S, S-Sepharose FF, Fractogel EMD SO₃M, Toyopearl MegacapII SP 550C, Poros 50 HS, and SP-sepharose matrix. In some embodiments,more than one cation exchange chromatography process can be employed onthe composition.

Mixed mode chromatography refers to a method that utilizes more than oneform of interaction between the stationary phase and analytes in orderto achieve their separation from impurities (e.g., process-relatedimpurities, such as host-cell proteins, DNA, and/or endogenous oradventitious viruses). Examples of suitable anion exchange matrices areknown in the art, and can include, but are not limited to, Capto Adhere,Sartobind Q, Natrix Q, Chromasorb Q, and Mustang Q.

In some embodiments, additional filtration steps can be used to removeimpurities. For example, in some embodiments nanofiltration orultrafiltration is used. Nanofiltration comprises passing thecomposition through a matrix having a pore size of, e.g., less than 75nm, less than 50 nm, and even less than 15 nm, to separate impurities,e.g., viruses, from the antibody. Commercially available nanofilters andultrafilters that can be employed are manufactured by various vendorssuch as Millipore Corporation (Billerica, Mass., e.g., Viresolve Pro andViresolve Pro+), Pall Corporation (East Hills, N.Y.), GE HealthcareSciences (Piscataway, N.J.), and Sartorius Corporation (Goettingen,Germany).

In some embodiments, the antibody of the present invention, e.g., ananti-IL5R comprising a heavy chain variable region and a light chainvariable region, wherein the heavy chain variable region comprises theKabat-defined CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 5-7, andwherein the light chain variable region comprises the Kabat-definedCDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 8-10 is at 1 mg/ml to 200mg/ml, 2 mg/ml to 100 mg/ml, 2 mg/ml to 30 mg/ml, 2 mg/ml to 25 mg/ml, 2mg/ml to 20 mg/ml, 3 mg/ml, 4 mg/ml, 5 mg/ml, 6 mg/ml, 7 mg/ml, 8 mg/ml,9 mg/ml, 10 mg/ml, 11 mg/ml, 12 mg/ml, 13 mg/ml, 14 mg/ml, 15 mg/ml, 16mg/ml, 17 mg/ml, 18 mg/ml, 19 mg/ml, or 20 mg/ml. In some embodiments,the antibody of the present invention, e.g., anti-IL5R, is concentrationof about 20 mg/ml, 25 mg/ml, 30 mg/ml, 40 mg/ml, 45 mg/ml, 50 mg/ml, 55mg/ml, 60 mg/ml, 65 mg/ml, 70 mg/ml, 75 mg/ml, 80 mg/ml, 85 mg/ml, 90mg/ml, 95 mg/ml, or 100 mg/ml.

The antibody formulation of the present invention can comprise anuncharged excipient. The term excipient refers to a pharmacologicallyinactive substance formulated with the antibody as described herein. Insome embodiments, the excipient can assist in the prevention ofdenaturation or otherwise assist in stabilizing the antibody. Suitableexcipients that may be used in the pharmaceutical compositions are knownin the art. Examples can be taken e.g. from the handbook: Gennaro,Alfonso R.: “Remington's Pharmaceutical Sciences”, Mack PublishingCompany, Easton, Pa., 1990. In some embodiments, the excipient is an“uncharged” excipient, i.e., the excipient does not carry either apositive “+” or negative “−” charge. In some embodiments, the excipientis selected from the group consisting of fructose, glucose, mannose,sorbose, xylose, lactose, maltose, sucrose, dextran, pullulan, dextrin,cyclodextrins, soluble starch, trehalose, sorbitol, erythritol, isomalt,lactitol, maltitol, xylitol, glycerol, lactitol, hydroxyethyl starch,water-soluble glucans.

In some embodiments, the uncharged excipient is about 1 mM to about 1 M,about 2 mM to about 500 mM, about 5 mM to about 400 mM, about 10 mM toabout 300 mM or about 20 mM to about 250 mM in the antibody formulation.In some embodiments, the uncharged excipient is about 5 mM to about 150mM, about 10 mM to about 100 mM, about 20 mM to about 80 mM, about 30mM, about 40 mM, about 50 mM, about 60 mM, or about 70 mM in theantibody formulation, e.g., an antibody formulation comprising 2 to 20mg/mL antibody. In one embodiment, the uncharged excipient is about 50mM in the antibody formulation. In some embodiments, the unchargedexcipient is about 50 mM to about 800 mM, about 100 mM to about 500 mM,about 150 mM to about 400 mM, about 200 mM, about 400 mM, about 200 mM,about 300 mM, or about 250 mM in the antibody formulation, e.g., anantibody formulation comprising 20 to 100 mg/mL antibody. In oneembodiment, the uncharged excipient is about 250 mM in the antibodyformulation.

In some embodiments, the uncharged excipient is trehalose, asrepresented by the formula:

In some embodiments, the trehalose is about 1 mM to about 1 M, about 2mM to about 500 mM, about 5 mM to about 400 mM, about 10 mM to about 300mM or about 20 mM to about 250 mM in the antibody formulation. In someembodiments, the trehalose is about 5 mM to about 150 mM, about 10 mM toabout 100 mM, about 20 mM to about 80 mM, about 30 mM, about 40 mM,about 50 mM, about 60 mM, or about 70 mM in the antibody formulation,e.g., an antibody formulation comprising 2 to 20 mg/mL antibody. In oneembodiment, the trehalose is about 50 mM in the antibody formulation. Insome embodiments, the trehalose is about 50 mM to about 800 mM, about100 mM to about 500 mM, about 150 mM to about 400 mM, about 200 mM,about 400 mM, about 200 mM, about 300 mM, or about 250 mM in theantibody formulation, e.g., an antibody formulation comprising 20 to 100mg/mL antibody. In one embodiment, the trehalose is about 250 mM in theantibody formulation.

The antibody formulation of the present invention comprises arginine.Arginine is a conditionally non-essential amino acid that can berepresented by the formula:

Arginine, as used herein, can include the free base form of arginine, aswell as any and all salts thereof. In some embodiments, arginineincludes a pharmaceutically acceptable salt thereof. For example,arginine would include arginine hydrochloride. Arginine, as used herein,also includes all enantiomers (e.g., L-arginine and S-arginine), and anycombination of enantiomers (e.g., 50% L-arginine and 50% S-arginine;90%-100% L-arginine and 10%-0% S-arginine, etc.). In some embodiments,the term “arginine” includes greater than 99% L-arginine and less than1% S-arginine. In some embodiments, the term “arginine” includes aenantomerically pure L-arginine. In some embodiments, arginine is apharmaceutical grade arginine.

Various concentrations of arginine can be present in the antibodyformulation. In some embodiments, the antibody formulation comprisesgreater than 50 mM arginine, greater than 75 mM arginine, greater than100 mM arginine, greater than 125 mM arginine, greater than 130 mMarginine, greater than 150 mM arginine, greater than 175 mM arginine, orgreater than 200 mM arginine.

In some embodiments, the antibody formulation comprises up to 800 mMarginine, up to 600 mM arginine, up to 400 mM arginine, up to 200 mMarginine, up to 150 mM arginine, up to 130 mM arginine, or up to 125 mMarginine. In some embodiments, the antibody formulation comprises 50 mMto 300 mM, 75 mM to 250 mM, 100 mM to 200 mM, 110 mM to 160 mM, 120 mMto 150 mM, or about 125 mM arginine. In some embodiments, the antibodyformulation comprises 125 mM arginine. In some embodiments, the antibodyformulation comprises 130 mM arginine. In some embodiments, arginine isadded in an amount sufficient to maintain osmolality of the formulation.In some embodiments, arginine is added in an amount sufficient toachieve a hyper-tonic solution. Applicants have found that in someembodiments, increased ionic strength of the antibody formulationsprovides increased stability and a reduction in particle formation.

The antibody formulations described herein can have various viscosities.Methods of measuring viscosity of antibody formulations are known tothose in the art, and can include, e.g., a rheometer (e.g., Anton PaarMCR301 Rheometer with either a 50 mm, 40 mm or 20 mm plate accessory).In some embodiments of the present invention, the viscosities werereported at a high shear limit of 1000 per second shear rate. In someembodiments, the antibody formulation has a viscosity of less than 20centipoise (cP), less than 18 cP, less than 15 cP, less than 13 cP, orless than 11 cP. In some embodiments, the antibody formulation has aviscosity of less than 13 cP. One of skill in the art will appreciatethat viscosity is dependent on temperature, thus, unless otherwisespecified, the viscosities provided herein are measured at 25° C. unlessotherwise specified.

The term “injection force” is the amount of pressure (in Newtons)required to pass the antibody formulation through a needle. Theinjection force is correlated with the amount of resistance provided bythe antibody formulation when administering the antibody formulation toa subject. The injection force will be dependent on the gauge of theadministering needle, as well as temperature. In some embodiments, theantibody formulation has an injection force of less than 15 N, 12 N, 10N, or 8 N when passed through a 27 Ga thin wall PFS needle. In someembodiments, the antibody formulation has an injection force of lessthan 15 N, 12 N, 10 N, or 8 N when passed through a 29 Ga thin wall PFSneedle.

The antibody formulations can have different osmolarity concentrations.Methods of measuring osmolarity of antibody formulations are known tothose in the art, and can include, e.g., an osmometer (e.g., an AdvancedInstrument Inc 2020 freezing point depression osmometer). In someembodiments, the formulation has an osmolarity of between 200 and 600mosm/kg, between 260 and 500 mosm/kg, or between 300 and 450 mosm/kg.

The antibody formulation of the present invention can have various pHlevels. In some embodiments, the pH of the antibody formulation isbetween 4 and 7, between 4.5 and 6.5, or between 5 and 6. In someembodiments, the pH of the antibody formulation is 5.0. In someembodiments, the pH of the antibody formulation is 6.0. In someembodiments, the pH of the antibody formulation is >7.0. Various meansmay be utilized in achieving the desired pH level, including, but notlimited to the addition of the appropriate buffer.

Various other components can be included in the antibody formulation. Insome embodiments, the antibody formulation can comprise a buffer (e.g.,histidine, acetate, phosphate or citrate buffer), a surfactant (e.g.polysorbate), and/or a stabilizer agent (e.g. human albumin), etc. Insome embodiments, the antibody formulation can comprise pharmaceuticallyacceptable carriers, including, e.g., ion exchangers, alumina, aluminumstearate, lecithin, serum proteins, such as human serum albumin, buffersubstances such as phosphates, sucrose, glycine, sorbic acid, potassiumsorbate, partial glyceride mixtures of saturated vegetable fatty acids,water, salts or electrolytes, such as protamine sulfate, disodiumhydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zincsalts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone,cellulose-based substances, polyethylene glycol, sodiumcarboxymethylcellulose, polyacrylates,polyethylene-polyoxypropylene-block polymers, and polyethylene glycol.In some embodiments, the antibody formulation further comprises asurfactant. In some embodiments, the surfactant is selected from thegroup consisting of polysorbate, sodium dodecyl sulfate, and nonionicsurfactant.

In some embodiments, the surfactant is polysorbate 20, i.e.,polyoxyethylene (20) sorbitan monolaurate, as represented by theformula:

Polysorbate 20 (PS-20) is available commercially from several commercialvendors, e.g., Alkest® TW 20 (Oxiteno, Brazil), and Tween® 20 (Pierce,Rockford Ill.). Applicants have found that by carefully controlling theconcentration of PS-20 in the antibody formulation, the antibody hasadded stability and has reduced amounts of particle formation whenstored for extended periods of time.

In some embodiments, PS-20 is about 0.001% to about 0.02%, about 0.002%to about 0.015%, about 0.002% to about 0.01%, about 0.004% to about0.009%, about 0.005% to about 0.008%, about 0.007%, or about 0.006% ofthe antibody formulation.

In some embodiments, the antibody formulation further compriseshistidine. In some embodiments, the antibody formulation comprises about1 mM to about 100 mM, about 5 mM to about 80 mM histidine, about 10 mMto about 60 mM histidine, about 15 mM to about 50 mM histidine, about 15mM to about 30 mM histidine, or about 20 mM histidine.

In some embodiments, various components can be omitted from the antibodyformulation, or can be “substantially free” of that component. The term“substantially free” as used herein refers to an antibody formulation,said formulation containing less than 0.01%, less than 0.001%, less than0.0005%, less than 0.0003%, or less than 0.0001% of the designatedcomponent.

In some embodiments, the antibody formulation is substantially free of asaccharide, i.e., the antibody formulation, said formulation containingless than 0.01%, less than 0.001%, less than 0.0005%, less than 0.0003%,or less than 0.0001% of a saccharide. The term “saccharide” as usedherein refers to a class of molecules that are derivatives of polyhydricalcohols. Saccharides are commonly referred to as carbohydrates and maycontain different amounts of sugar (saccharide) units, e.g.,monosaccharides, disaccharides and polysaccharides. In some embodiments,the formulation is substantially free of disaccharide. In someembodiments, the formulation substantially free of a reducing sugar, anon-reducing sugar, or a sugar alcohol. In some embodiments, theantibody formulation is substantially free of proline, glutamate,sorbitol, divalent metal ions, and/or succinate.

In some embodiments, the invention is directed to a stable, aqueousantibody formulation comprising: (a) about 2 mg/mL to about 20 mg/mL ofan antibody, wherein the antibody comprises a heavy chain variableregion and a light chain variable region, wherein the heavy chainvariable region comprises the Kabat-defined CDR1, CDR2, and CDR3sequences of SEQ ID NOs: 5-7, and wherein the light chain variableregion comprises the Kabat-defined CDR1, CDR2, and CDR3 sequences of SEQID NOs: 8-10, (b) about 0.002% to about 0.01% polysorbate-20, (c) about40 mM to about 60 mM trehalose, and (d) about 110 mM to about 150 mML-arginine. In some embodiments, the formulation further comprises about20 mM histidine. In one embodiment, the invention is directed to astable, aqueous antibody formulation comprising: (a) about 2 mg/mL toabout 20 mg/mL of an antibody, wherein the antibody comprises a heavychain variable region and a light chain variable region, wherein theheavy chain variable region comprises the Kabat-defined CDR1, CDR2, andCDR3 sequences of SEQ ID NOs: 5-7, and wherein the light chain variableregion comprises the Kabat-defined CDR1, CDR2, and CDR3 sequences of SEQID NOs: 8-10, (b) about 0.006% polysorbate-20, (c) about 50 mMtrehalose, (d) about 130 mM L-arginine, and (e) about 20 mM histidine.

In some embodiments, the invention is directed to a stable, aqueousantibody formulation comprising: (a) about 20 mg/mL to about 100 mg/mLof an antibody, wherein the antibody comprises a heavy chain variableregion and a light chain variable region, wherein the heavy chainvariable region comprises the Kabat-defined CDR1, CDR2, and CDR3sequences of SEQ ID NOs: 5-7, and wherein the light chain variableregion comprises the Kabat-defined CDR1, CDR2, and CDR3 sequences of SEQID NOs: 8-10, (b) about 0.002% to about 0.01% polysorbate-20, (c) about200 mM to about 300 mM trehalose, and (d) about 20 mM histidine. In oneembodiment, the invention is directed to a stable, aqueous antibodyformulation comprising: (a) about 20 mg/mL to about 100 mg/mL of anantibody, wherein the antibody comprises a heavy chain variable regionand a light chain variable region, wherein the heavy chain variableregion comprises the Kabat-defined CDR1, CDR2, and CDR3 sequences of SEQID NOs: 5-7, and wherein the light chain variable region comprises theKabat-defined CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 8-10, (b)about 0.006% polysorbate-20, (c) about 250 mM trehalose, and (d) about20 mM histidine. In another embodiment, the invention is directed to astable, aqueous antibody formulation comprising: (a) about 30 mg/mL ofan antibody, wherein the antibody comprises a heavy chain variableregion and a light chain variable region, wherein the heavy chainvariable region comprises the Kabat-defined CDR1, CDR2, and CDR3sequences of SEQ ID NOs: 5-7, and wherein the light chain variableregion comprises the Kabat-defined CDR1, CDR2, and CDR3 sequences of SEQID NOs: 8-10, (b) about 0.006% polysorbate-20, (c) about 250 mMtrehalose, and (d) about 20 mM histidine.

The antibody formulations of the present invention are an aqueoussolution. In some embodiments, the antibody formulation has not beensubjected to freezing temperatures, and/or have not been frozen, i.e.,they have remained in a liquid state. In some embodiments, the antibodyin the antibody formulation has not been subjected to lyophilization.

As used herein, the term “stability” generally is related to maintainingthe integrity or to minimizing the degradation, denaturation,aggregation or unfolding of a biologically active agent such as aprotein, peptide or another bioactive macromolecule. As used herein,“improved stability” generally means that, under conditions known toresult in degradation, denaturation, aggregation or unfolding, theprotein (e.g., antibody such as anti-IL5R), peptide or another bioactivemacromolecule of interest maintains greater stability compared to acontrol protein, peptide or another bioactive macromolecule.

In some embodiments, stability refers to an antibody formulation havinglow to undetectable levels of particle formation. The phrase “low toundetectable levels of particle formation” as used herein refers tosamples containing less than 30 particles/mL, less than 20 particles/ml,less than 20 particles/ml, less than 15 particles/ml, less than 10particles/ml, less than 5 particles/ml, less than 2 particles/ml or lessthan 1 particle/ml as determined by HIAC analysis or visual analysis. Insome embodiments, no particles in the antibody formulation are detected,either by HIAC analysis or visual analysis.

In some embodiments, stability refers to reduced fragmentation of theantibody. The term “low to undetectable levels of fragmentation” as usedherein refers to samples containing equal to or more than 80%, 85%, 90%,95%, 98% or 99% of the total protein, for example, in a single peak asdetermined by HPSEC, or in two peaks (e.g., heavy- and light-chains) (oras many peaks as there are subunits) by reduced Capillary GelElectrophoresis (rCGE), representing the non-degraded antibody or anon-degraded fragment thereof, and containing no other single peakshaving more than 5%, more than 4%, more than 3%, more than 2%, more than1%, or more than 0.5% of the total protein in each. The term “reducedCapillary Gel Electrophoresis” as used herein refers to capillary gelelectrophoresis under reducing conditions sufficient to reduce disulfidebonds in an antibody.

One of skill in the art will appreciate that stability of a protein isdependent on other features in addition to the composition of theformulation. For example, stability can be affected by temperature,pressure, humidity, pH, and external forms of radiation. Thus, unlessotherwise specified, stability referred to herein is considered to bemeasured at 5° C., one atmosphere pressure, 50% relative humidity, pH of6.0, and normal background levels of radiation. Stability of theantibody in the antibody formulation can be determined by various means.In some embodiments, the antibody stability is determined by sizeexclusion chromatography (SEC). SEC separates analytes (e.g.,macromolecules such as proteins and antibodies) on the basis of acombination of their hydrodynamic size, diffusion coefficient, andsurface properties. Thus, for example, SEC can separate antibodies intheir natural three-dimensional conformation from antibodies in variousstates of denaturation, and/or antibodies that have been degraded. InSEC, the stationary phase is generally composed of inert particlespacked into a dense three-dimensional matrix within a glass or steelcolumn. The mobile phase can be pure water, an aqueous buffer, anorganic solvent, mixtures of these, or other solvents. Thestationary-phase particles have small pores and/or channels which willonly allow species below a certain size to enter. Large particles aretherefore excluded from these pores and channels, but the smallerparticles are removed from the flowing mobile phase. The time particlesspend immobilized in the stationary-phase pores depends, in part, on howfar into the pores they can penetrate. Their removal from the mobilephase flow causes them to take longer to elute from the column andresults in a separation between the particles based on differences intheir size.

In some embodiments, SEC is combined with an identification technique toidentify or characterize proteins, or fragments thereof. Proteinidentification and characterization can be accomplished by varioustechniques, including but not limited chromatographic techniques, e.g.,high-performance liquid chromatography (HPLC), immunoassays,electrophoresis, ultra-violet/visible/infrared spectroscopy, ramanspectroscopy, surface enhanced raman spectroscopy, mass spectroscopy,gas chromatography, static light scattering (SLS), Fourier TransformInfrared Spectroscopy (FTIR), circular dichroism (CD), urea-inducedprotein unfolding techniques, intrinsic tryptophan fluorescence,differential scanning calorimetry, and/or ANS protein binding.

In some embodiments, protein identification is achieved by high-pressureliquid chromatography. Various instruments, and apparatuses are known tothose of skill in the art to perform HPLC. Generally HPLC involvesloading a liquid solvent containing the protein of interest onto aseparation column, in which the separation occurs. The HPLC separationcolumn is filled with solid particles (e.g. silica, polymers, orsorbents), and the sample mixture is separated into compounds as itinteracts with the column particles. HPLC separation is influenced bythe liquid solvent's condition (e.g. pressure, temperature), chemicalinteractions between the sample mixture and the liquid solvent (e.g.hydrophobicity, protonation, etc.), and chemical interactions betweenthe sample mixture and the solid particles packed inside of theseparation column (e.g. ligand affinity, ion exchange, etc.).

In some embodiments, the SEC and protein identification occurs withinthe same apparatus, or simultaneously. For example, SEC and HPLC can becombined, often referred to as SE-HPLC.

In some embodiments, the aqueous formulation comprises about 2 mg/ml toabout 100 mg/ml antibody wherein the antibody comprises a heavy chainvariable region and a light chain variable region, wherein the heavychain variable region comprises the Kabat-defined CDR1, CDR2, and CDR3sequences of SEQ ID NOs: 5-7, and wherein the light chain variableregion comprises the Kabat-defined CDR1, CDR2, and CDR3 sequences of SEQID NOs: 8-10, wherein said formulation is stable upon storage at about40° C. for at least 1 month. In some embodiments, the formulation isstable upon storage at about 25° C. for at least 3 months. In someembodiments, the formulation is stable upon storage at about 5° C. forat least 6 months. In some embodiments, the formulation is stable uponstorage at about 5° C. for at least 12 months. In some embodiments, theformulation is stable upon storage at about 5° C. for at least 18months. In some embodiments, the formulation is stable upon storage atabout 5° C. for at least 24 months, or 36 months.

The term “stable” can be relative and not absolute. Thus, in someembodiments the antibody is stable if less than 20%, less than 15%, lessthan 10%, less than 5% or less than 2% of the antibody is degraded,denatured, aggregated or unfolded as determined by SEC HPLC when theantibody is stored at 2° C. to 8° C. for 6 months. In some embodiments,the antibody is stable if less than 20%, less than 15%, less than 10%,less than 5% or less than 2% of the antibody is degraded, denatured,aggregated or unfolded as determined by SEC HPLC when the antibody isstored at 2° C. to 8° C. for 12 months. In some embodiments, theantibody in the antibody formulation is stable if less than 20%, lessthan 15%, less than 10%, less than 5% or less than 2% of the antibody isdegraded, denatured, aggregated or unfolded as determined by SEC HPLCwhen the antibody is stored at 2° C. to 8° C. for 18 months. In someembodiments, the antibody in the antibody formulation is stable if lessthan 20%, less than 15%, less than 10%, less than 5% or less than 2% ofthe antibody is degraded, denatured, aggregated or unfolded asdetermined by SEC HPLC when the antibody is stored at 2° C. to 8° C. for24 months.

In some embodiments, the antibody is stable if less than 20%, less than15%, less than 10%, less than 5% or less than 2% of the antibody isdegraded, denatured, aggregated or unfolded as determined by SEC HPLCwhen the antibody is stored at 23° C. to 27° C. for 3 months. In someembodiments, the antibody is stable if less than 20%, less than 15%,less than 10%, less than 5% or less than 2% of the antibody is degraded,denatured, aggregated or unfolded as determined by SEC HPLC when theantibody is stored at 23° C. to 27° C. for 6 months. In someembodiments, the antibody is stable if less than 20%, less than 15%,less than 10%, less than 5% or less than 2% of the antibody is degraded,denatured, aggregated or unfolded as determined by SEC HPLC when theantibody is stored at 23° C. to 27° C. for 12 months. In someembodiments, the antibody is stable if less than 20%, less than 15%,less than 10%, less than 5% or less than 2% of the antibody is degraded,denatured, aggregated or unfolded as determined by SEC HPLC when theantibody is stored at 23° C. to 27° C. for 24 months.

In some embodiments the antibody is stable if less than 6%, less than4%, less than 3%, less than 2% or less than 1% of the antibody isdegraded, denatured, aggregated or unfolded per month as determined bySEC HPLC when the antibody is stored at 40° C. In some embodiments theantibody is stable if less than 6%, less than 4%, less than 3%, lessthan 2% or less than 1% of the antibody is degraded, denatured,aggregated or unfolded per month as determined by SEC HPLC when theantibody is stored at 5° C.

In some embodiments, the antibody formulations of the present inventioncan be considered stable if the antibody exhibits very little to no lossof the binding activity of the antibody (including antibody fragmentsthereof) of the formulation compared to a reference antibody as measuredby antibody binding assays know to those in the art, such as, e.g.,ELISAs, etc., over a period of 8 weeks, 4 months, 6 months, 9 months, 12months or 24 months. In some embodiments, the antibody stored at about40° C. for at least 1 month retains at least 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 98%, or at leastabout 99% of binding ability to an IL-5 receptor polypeptide compared toa reference antibody which has not been stored. In some embodiments, theantibody stored at about 5° C. for at least 6 months retains at least80%, at least about 85%, at least about 90%, at least about 95%, atleast about 98%, or at least about 99% of binding ability to an IL-5receptor polypeptide compared to a reference antibody which has not beenstored. In some embodiments, the antibody stored at about 40° C. for atleast 1 month retains at least 95% of binding ability to an IL-5receptor polypeptide compared to a reference antibody which has not beenstored. In some embodiments, the antibody stored at about 5° C. for atleast 6 months retains at least 95% of binding ability to an IL-5receptor polypeptide compared to a reference antibody which has not beenstored.

The antibody formulations can provide low to undetectable levels ofaggregation of the antibody. The phrase “low to undetectable levels ofaggregation” as used herein refers to samples containing no more thanabout 5%, no more than about 4%, no more than about 3%, no more thanabout 2%, no more than about 1% and no more than about 0.5% aggregationby weight of protein as measured by high performance size exclusionchromatography (HPSEC) or static light scattering (SLS) techniques. Insome embodiments, less than 2% of the antibody forms an aggregate uponstorage at about 40° C. for at least 4 weeks as determined by asdetermined by HPSEC. In some embodiments, less than 2% of the antibodyforms an aggregate upon storage at about 5° for at least 3 months, atleast 6 months, at least 9 months, at least 12 months, at least 15months, at least 18 months, at least 24 months, or at least 36 months asdetermined by HPSEC.

Applicants have found the antibody formulations provided herein resultin greatly reduced particle formation as determined by visualinspection, micro-flow imaging (MFI), or size-exclusion chromatography(SEC). In some embodiments, the formulation is substantially free ofparticles upon storage at about 40° C. for at least 1 month asdetermined by visual inspection. In some embodiments, the formulation issubstantially free from particles upon storage at about 5° C. for atleast 6 months, at least 9 months, at least 12 months, at least 15months, at least 18 months, at least 24 months, or at least 36 months asdetermined by visual inspection.

In some embodiments, the antibody formulation of the present inventioncan be used for pharmaceutical purposes. Antibodies used inpharmaceutical applications generally must have a high level of purity,especially in regard to contaminants from the cell culture, includingcellular protein contaminants, cellular DNA contaminants, viruses andother transmissible agents. See “WHO Requirements for the use of animalcells as in vitro substrates for the production of biologicals:Requirements for Biological Substances No. 50.” No. 878. Annex 1, 1998.In response to concerns about contaminants, The World HealthOrganization (WHO) established limits on the levels of variouscontaminants. For example, the WHO recommended a DNA limit of less than10 ng per dose for protein products. Likewise, the United States Foodand Drug Administration (FDA) set a DNA limit of less than or equal to0.5 μg/mg protein. Thus, in some embodiments, the present invention isdirected to antibody formulations meeting or exceeding contaminantlimits as defined by one or more governmental organizations, e.g., theUnited States Food and Drug Administration and/or the World HealthOrganization.

In some embodiments, the antibody formulation described herein ispharmaceutically acceptable. “Pharmaceutically acceptable” refers to anantibody formulation that is, within the scope of sound medicaljudgment, suitable for contact with the tissues of human beings andanimals without excessive toxicity or other complications commensuratewith a reasonable benefit/risk ratio.

Purity of the antibody formulations can vary. In some embodiments, thetherapeutic antibody of interest, e.g., anti-IL5R antibody, is greaterthan 90% (wt/wt) of the total polypeptides present in the antibodyformulation. In some embodiments, the therapeutic antibody of interest,e.g., anti-IL5R is greater than 95% (wt/wt), 98% (wt/wt), 99% (wt/wt),99.5% (wt/wt) or 99.9% (wt/wt) of the total polypeptide present in theantibody formulation.

The formulations as provided herein can be suitable for treatment of asubject. As used herein, “subject” can be used interchangeably with“patient” and refers to any animal classified as a mammal, includinghumans and non-humans, such as, but not limited to, domestic and farmanimals, zoo animals, sports animals, and pets. In some embodiments,subject refers to a human.

The terms “treat” and “treatment” refer to both therapeutic treatmentand prophylactic, maintenance, or preventative measures, wherein theobject is to prevent or alleviate (lessen) an undesired physiologicalcondition, disorder or disease, or obtain beneficial or desired clinicalresults. The terms “treat,” “treatment,” and “treating” refer to thereduction or amelioration of the progression, severity, and/or durationof such a disease or disorder (e.g., a disease or disorder characterizedby aberrant expression and/or activity of an IL-5 polypeptide, a diseaseor disorder characterized by aberrant expression and/or activity of anIL-5 polypeptide or one or more subunits thereof, an autoimmune disease,an inflammatory disease, a proliferative disease, or an infection) orthe amelioration of one or more symptoms thereof resulting from theadministration of one or more therapies (including, but not limited to,the administration of one or more prophylactic or therapeutic agents).In certain embodiments, such terms refer to reduction in inflammationassociated eosinophil-mediated inflammation. In other embodiments, suchterms refer to the reduction of the release of inflammatory agents bymast cells, or the reduction of the biological effect of suchinflammatory agents. In other embodiments, such terms refer to areduction of the growth, formation and/or increase in the number ofhyperproliferative cells (e.g., cancerous cells). In yet otherembodiments, such terms refer to the reduction of inflammation of theairways, skin, gastrointestinal tract, or combinations thereof. In yetother embodiments, such terms refer to the reduction in the symptomsassociated with asthma. In some embodiments, such terms refer to thereduction in the symptoms associate with chronic obstructive pulmonarydisease (COPD).

The antibody formulation of the present invention can be administered toa subject through various means. In some embodiments, the antibodyformulation is suitable for parenteral administration, e.g., viainhalation (e.g., powder or aerosol spray), transmucosal, intravenous,subcutaneous, or intramuscular administration. In some embodiments, theformulation is an injectable formulation. In some embodiments, theinvention is directed to a sealed container comprising any of theantibody formulations as described herein.

In some aspects, the present invention is directed to variouspharmaceutical dosage forms. Various dosage forms could be applicable tothe formulations provided herein. See, e.g., Pharmaceutical Dosage Form:Parenteral Medications, Volume 1, 2^(nd) Edition. In one embodiment, apharmaceutical unit dosage of the invention comprises the antibodyformulation in a suitable container, e.g. a vial or syringe. In oneembodiment, a pharmaceutical unit dosage of the invention comprises anintravenously, subcutaneously, or intramuscularly delivered antibodyformulation. In another embodiment, a pharmaceutical unit dosage of theinvention comprises aerosol delivered antibody formulation. In aspecific embodiment, a pharmaceutical unit dosage of the inventioncomprises a subcutaneously delivered antibody formulation. In anotherembodiment, a pharmaceutical unit dosage of the invention comprises anaerosol delivered antibody formulation. In a further embodiment, apharmaceutical unit dosage of the invention comprises an intranasallyadministered antibody formulation.

The antibody formulations of the present invention can be prepared asunit dosage forms by preparing a vial containing an aliquot of theaqueous antibody formulation for a one-time use. For example, a unitdosage per vial may contain 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8ml, 9 ml, 10 ml, 15 ml, or 20 ml of different concentrations of anantibody that specifically binds to IL5 receptor ranging from about 0.1mg/ml to about 300 mg/ml. If necessary, these preparations can beadjusted to a desired concentration by adding a sterile diluent to eachvial. In a specific embodiment, the aqueous antibody formulations of thepresent invention are formulated into single dose vials as a sterileliquid that contains about 2 mg/mL to about 20 mg/mL of an antibody,wherein the antibody comprises a heavy chain variable region and a lightchain variable region, wherein the heavy chain variable region comprisesthe Kabat-defined CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 5-7, andwherein the light chain variable region comprises the Kabat-definedCDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 8-10, about 0.002% toabout 0.01% polysorbate-20, about 40 mM to about 60 mM trehalose, andabout 110 mM to about 150 mM L-arginine. In another specific embodiment,the aqueous antibody formulations of the present invention areformulated into single dose vials as a sterile liquid that containsabout 20 mg/mL to about 100 mg/mL of an antibody, wherein the antibodycomprises a heavy chain variable region and a light chain variableregion, wherein the heavy chain variable region comprises theKabat-defined CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 5-7, andwherein the light chain variable region comprises the Kabat-definedCDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 8-10, about 0.002% toabout 0.01% polysorbate-20, and about 200 mM to about 300 mM trehalose.In one embodiment, the antibody of the invention is supplied at 2 to 20mg/ml in 3 cc USP Type I borosilicate amber vials (West PharmaceuticalServices-Part No. 6800-0675). In another embodiment, the antibody of theinvention is supplied at 20 to 100 mg/ml in 3 cc USP Type I borosilicateamber vials. The target fill volume is 1.2 mL.

The antibody formulations of the present invention can be prepared asunit dosage forms by preparing a pre-filled syringe containing analiquot of the aqueous antibody formulation for a one-time use. Forexample, a unit dosage per pre-filled syringe may contain 0.1 ml, 0.2ml, 0.3 ml, 0.4 ml, 0.5 ml, 0.6 ml, 0.7 ml, 0.8 ml, 0.9 ml, 1 ml, 2 ml,3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, 15 ml, or 20 ml ofdifferent concentrations of an antibody that specifically binds to anIL-5 polypeptide ranging from about 2 mg/ml to about 100 mg/ml. In aspecific embodiment, the aqueous antibody formulations of the presentinvention are formulated into single dose pre-filled syringes as asterile liquid that contains about 2 mg/mL to about 20 mg/mL of anantibody, wherein the antibody comprises a heavy chain variable regionand a light chain variable region, wherein the heavy chain variableregion comprises the Kabat-defined CDR1, CDR2, and CDR3 sequences of SEQID NOs: 5-7, and wherein the light chain variable region comprises theKabat-defined CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 8-10, about0.002% to about 0.01% polysorbate-20, about 40 mM to about 60 mMtrehalose, and about 110 mM to about 150 mM L-arginine. In a specificembodiment, the aqueous antibody formulations of the present inventionare formulated into single dose pre-filled syringes as a sterile liquidthat contains about 20 mg/mL to about 100 mg/mL of an antibody, whereinthe antibody comprises a heavy chain variable region and a light chainvariable region, wherein the heavy chain variable region comprises theKabat-defined CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 5-7, andwherein the light chain variable region comprises the Kabat-definedCDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 8-10, about 0.002% toabout 0.01% polysorbate-20, and about 200 mM to about 300 mM trehalose.

Various dosage amounts can be administered in a single use. For example,in some embodiments 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7mg, 0.8 mg, 0.9 mg, 1.0 mg, 1.1 mg, 1.2 mg, 1.3 mg, 1.4 mg, 1.5 mg, 1.6mg, 1.7 mg, 1.8 mg, 1.9 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9mg, 10 mg, 12 mg, 14 mg, 16 mg, 18 mg, 20 mg, 30 mg, 40 mg, 50 mg, 70mg, or 100 mg of antibody can be administered in a single dose.

Various types of syringes can be used. The syringe can be filled withthe antibody formulation immediately prior to administration to asubject, e.g., less than 1 week, 1 day, 6 hours, 3 hours, 2 hours, 1hour, 30 minutes, 20 minutes, or 10 minutes prior to administration to asubject. In some embodiments, the syringe is filled with the antibodyformulation at the point of retail sale, or by the facility for whichtreatment of the subject occurs. In some embodiments, the syringe ispre-filled, e.g., the syringe is filled with the antibody formulationgreater than 1 day, 2 days, 4 days, 1 week, 2 weeks, 1 month, 2 months,3 months, 6 months, 12 months, 18 months, 24 months, 3 years, or 4 yearsprior to administration to a subject. In some embodiments, thepre-filled syringe comprises a needle, e.g., a 27 G regular wall needle,a 27 G thin wall needle, a 29 G regular wall needle, or a 29 G thin wallneedle. In some embodiments, the pre-filled syringe comprises a 29 Gthin wall needle.

In some embodiments, any syringe suitable for administration to thedesired subject can be used. In some embodiments, the syringe is aplastic syringe or a glass syringe. In some embodiments, the syringe ismade of materials that are substantially free from tungsten. In someembodiments, the syringe is coated with silicone. In some embodiments,the pre-filled syringe comprises a plunger having a fluoropolymer resindisk. Examples of syringes can include, but are not limited to Hypak™for Biotech 1 ml Long (Becton Dickinson), with a Becton Dickinson Hypak1 mL long plunger stopper 4023 Flurotec Daikyo Sil000 (Catalog#47271919), C3Pin (lot # E912701), Hypak™ for Biotech 0.8 mg siliconeoil (Becton Dickinson), and CZ syringes (West, Catalog #19550807).

The aqueous antibody formulations of the present invention may besterilized by various sterilization methods, including sterilefiltration, radiation, etc. In a specific embodiment, the difilteredantibody formulation is filter-sterilized with a presterilized 0.2micron filter. Sterilized aqueous antibody formulations of the presentinvention may be administered to a subject to prevent, treat and/ormanage an immune response, e.g. an inflammatory response.

In some embodiments, the pre-filled syringe comprises (a) about 2 mg/mLto about 20 mg/mL of an antibody, wherein the antibody comprises a heavychain variable region and a light chain variable region, wherein theheavy chain variable region comprises the Kabat-defined CDR1, CDR2, andCDR3 sequences of SEQ ID NOs: 5-7, and wherein the light chain variableregion comprises the Kabat-defined CDR1, CDR2, and CDR3 sequences of SEQID NOs: 8-10, and (b) about 0.002% to about 0.01% polysorbate-20. Insome embodiments, the pre-filled syringe further comprises (c) about 40mM to about 60 mM trehalose, and (d) about 110 mM to about 150 mML-arginine. In some embodiments, the pre-filled syringe comprises (a)about 20 mg/mL to about 100 mg/mL of an antibody, wherein the antibodycomprises a heavy chain variable region and a light chain variableregion, wherein the heavy chain variable region comprises theKabat-defined CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 5-7, andwherein the light chain variable region comprises the Kabat-definedCDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 8-10, and (b) about 0.002%to about 0.01% polysorbate-20. In some embodiments, the pre-filledsyringe further comprises (c) about 200 mM to about 300 mM trehalose.

In some embodiments, the invention is directed to a kit comprising anyof the antibody formulations described herein, the containers describedherein, the unit dosage forms described herein, or the pre-filledsyringe described herein.

In some embodiments, the present invention can also be directed to amethod of producing a stable, aqueous antibody formulation comprising anantibody, the method comprising: (a) purifying an antibody to about 1mg/mL to about 400 mg/mL, wherein the antibody comprises a heavy chainvariable region and a light chain variable region, wherein the heavychain variable region comprises the Kabat-defined CDR1, CDR2, and CDR3sequences of SEQ ID NOs: 5-7, and wherein the light chain variableregion comprises the Kabat-defined CDR1, CDR2, and CDR3 sequences of SEQID NOs: 8-10; and (b) placing the isolated antibody in a stabilizingformulation to form the stable, aqueous antibody formulation, whereinthe resulting stable, aqueous antibody formulation comprises: (i) about2 mg/mL to about 100 mg/mL of an antibody, wherein the antibodycomprises a heavy chain variable region and a light chain variableregion, wherein the heavy chain variable region comprises theKabat-defined CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 5-7, andwherein the light chain variable region comprises the Kabat-definedCDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 8-10, and (ii) about0.002% to about 0.01% polysorbate-20. In some embodiments, the inventionis directed to a method of making a stable, aqueous antibodyformulation, the method comprising: (a) purifying an antibody to about 1mg/mL to about 400 mg/mL, wherein the antibody comprises a heavy chainvariable region and a light chain variable region, wherein the heavychain variable region comprises the Kabat-defined CDR1, CDR2, and CDR3sequences of SEQ ID NOs: 5-7, and wherein the light chain variableregion comprises the Kabat-defined CDR1, CDR2, and CDR3 sequences of SEQID NOs: 8-10; (b) diluting the antibody to about 2 mg/mL to about 20mg/mL of an antibody, wherein the antibody comprises a heavy chainvariable region and a light chain variable region, wherein the heavychain variable region comprises the Kabat-defined CDR1, CDR2, and CDR3sequences of SEQ ID NOs: 5-7, and wherein the light chain variableregion comprises the Kabat-defined CDR1, CDR2, and CDR3 sequences of SEQID NOs: 8-10, into a solution comprising: (i) about 0.002% to about0.01% polysorbate-20, (ii) about 40 mM to about 60 mM trehalose, and(iii) about 110 mM to about 150 mM L-arginine. In some embodiments, theinvention is directed to a method of making a stable, aqueous antibodyformulation, the method comprising: (a) purifying an antibody to about 1mg/mL to about 400 mg/mL, wherein the antibody comprises a heavy chainvariable region and a light chain variable region, wherein the heavychain variable region comprises the Kabat-defined CDR1, CDR2, and CDR3sequences of SEQ ID NOs: 5-7, and wherein the light chain variableregion comprises the Kabat-defined CDR1, CDR2, and CDR3 sequences of SEQID NOs: 8-10; (b) diluting the antibody to about 20 mg/mL to about 100mg/mL of an antibody, wherein the antibody comprises a heavy chainvariable region and a light chain variable region, wherein the heavychain variable region comprises the Kabat-defined CDR1, CDR2, and CDR3sequences of SEQ ID NOs: 5-7, and wherein the light chain variableregion comprises the Kabat-defined CDR1, CDR2, and CDR3 sequences of SEQID NOs: 8-10, into a solution comprising: (i) about 0.002% to about0.01% polysorbate-20, (ii) about 200 mM to about 300 mM trehalose, and(iii) about 20 mM histidine.

Although many aspects of the invention are directed to aqueousformulations, it should be noted for the purpose of equivalents that theantibodies or antibody formulations of the invention may be lyophilizedif desired. Thus, the invention encompasses lyophilized forms of theformulations of the invention, or lyophilized antibodies which are laterreconstituted into an aqueous form. In some embodiments, the inventionis directed to a method of producing a reconstituted antibodyformulation comprising a heavy chain variable region and a light chainvariable region, wherein the heavy chain variable region comprises theKabat-defined CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 5-7, andwherein the light chain variable region comprises the Kabat-definedCDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 8-10, the methodcomprising: (a) purifying the antibody from a cell culture; (b)lyophilizing the isolated antibody; (c) adding the lyophilized antibodyto a aqueous solution to form a reconstituted antibody formulation,wherein the reconstituted antibody formulation comprises: (i) about 2mg/mL to about 100 mg/mL of an antibody, wherein the antibody comprisesa heavy chain variable region and a light chain variable region, whereinthe heavy chain variable region comprises the Kabat-defined CDR1, CDR2,and CDR3 sequences of SEQ ID NOs: 5-7, and wherein the light chainvariable region comprises the Kabat-defined CDR1, CDR2, and CDR3sequences of SEQ ID NOs: 8-10, and (ii) about 0.002% to about 0.01%polysorbate-20.

In some embodiments, the inventors have found that anti-IL5R antibodyformulations having decreased glutathione S-transferase (GST)concentrations result in reduced (e.g., non-detectable) particleformation. Removal of particles is important for avoiding potentialimmunogenicity as well as limiting impact on product quality. In someembodiments, the GST concentrations are reduced by affinitychromatography. In some embodiments, the GST concentrations are reducedby using a Protein A column. In some embodiments, the Protein A columnis MabSelect Sure (GE Healthcare Life Sciences). In some embodiments,the GST concentrations are reduced by using mixed mode chromatography.In some embodiments, the mixed mode column is Capto™ Adhere (GEHealthcare Life Sciences).

In some embodiments, the invention is directed to an antibodyformulation comprising an antibody wherein the antibody comprises aheavy chain variable region and a light chain variable region, whereinthe heavy chain variable region comprises the Kabat-defined CDR1, CDR2,and CDR3 sequences of SEQ ID NOs: 5-7, and wherein the light chainvariable region comprises the Kabat-defined CDR1, CDR2, and CDR3sequences of SEQ ID NOs: 8-10, wherein the antibody formulation isessentially free of particles. In some embodiments, the term“essentially free of particles” refer to the absence of visibleparticles when viewed under a light box. In some embodiments, the term“essentially free of particles” is synonymous with the phrase “low toundetectable levels of particle formation” as described previously. Insome embodiments, essentially free of particles refers to samplescontaining less than 30 particles/mL, less than 20 particles/ml, lessthan 20 particles/ml, less than 15 particles/ml, less than 10particles/ml, less than 5 particles/ml, less than 2 particles/ml or lessthan 1 particle/ml wherein the particles are greater than 25 μm and theparticle count is determined by HIAC analysis or visual analysis. Insome embodiments, essentially free of particles refers to samplescontaining 1 to 50 particles/mL, 2 to 40 particles/ml, 3-30particles/ml, 4 to 25 particles/ml, or 5 to 20 particles/ml wherein theparticles are greater than 25 μm and the particle count is determined byHIAC analysis or visual analysis. In some embodiments, the term “visibleparticles” refers to particles greater than 25 μm.

In some embodiments, essentially free of particles refers to samplescontaining 1 to 200 particles/mL, 10 to 150 particles/ml, 30-100particles/ml, or 40 to 80 particles/ml, wherein the particles aregreater than 5 μm and the particle count is determined by HIAC analysisor visual analysis. In some embodiments, the term “visible particles”refers to particles greater than 5 μm. In some embodiments, no particlesin the antibody formulation are detected, either by HIAC analysis orvisual analysis.

In some embodiments, the invention is directed to an antibodyformulation comprising an antibody wherein the antibody comprises aheavy chain variable region and a light chain variable region, whereinthe heavy chain variable region comprises the Kabat-defined CDR1, CDR2,and CDR3 sequences of SEQ ID NOs: 5-7, and wherein the light chainvariable region comprises the Kabat-defined CDR1, CDR2, and CDR3sequences of SEQ ID NOs: 8-10, wherein the antibody formulation isessentially free of glutathione S-transferase (GST). Unless specifiedotherwise, the term “essentially free of glutathione S-transferase” or“essentially free of GST” would encompass a composition lacking activeGST (but which could contain inactive GST) as well as a compositionwhich does not have the GST protein, either in active form or inactiveform. In some embodiments, the invention is directed to an antibodyformulation comprising an antibody wherein the antibody comprises aheavy chain variable region and a light chain variable region, whereinthe heavy chain variable region comprises the Kabat-defined CDR1, CDR2,and CDR3 sequences of SEQ ID NOs: 5-7, and wherein the light chainvariable region comprises the Kabat-defined CDR1, CDR2, and CDR3sequences of SEQ ID NOs: 8-10, wherein the antibody formulation isessentially free of active GST. The term “active GST” refers to GSTcapable of catalyzing the formation of the thiol group of glutathione(GSH) to an electrophilic compound such as 1-chloro-2,4-dinitrobenzene(CDNB) for form a GS-DNB conjugate. GST or glutathione S-transferaserefers to a family of enzymes that are capable of catalyzing numerousreactions, but mainly the conjugation of a reduced glutathione, via asulfhydryl group, to electrophilic centers, e.g., aromatics, doublebonds, C—Cl_(x), etc. GST monomers are generally in the range of 22-29kDa, but they can occur as dimers, trimers, and also hetero-dimers (withother proteins). In some embodiments, the term GST refers to a proteinthat is capable of catalyzing the formation of the thiol group ofglutathione (GSH) to 1-chloro-2,4-dinitrobenzene (CDNB) for form aGS-DNB conjugate.

In some embodiments, the invention is directed to an antibodyformulation comprising an antibody wherein the antibody comprises aheavy chain variable region and a light chain variable region, whereinthe heavy chain variable region comprises the Kabat-defined CDR1, CDR2,and CDR3 sequences of SEQ ID NOs: 5-7, and wherein the light chainvariable region comprises the Kabat-defined CDR1, CDR2, and CDR3sequences of SEQ ID NOs: 8-10, wherein the antibody formulation isessentially free of particles for at least 1 month, at least 2 months,at least 3 months, at least 4 months, at least 5 months, at least 6months, at least 8 months, at least 10 months, at least 12 months, or atleast 18 months when stored at 38-42° C. In some embodiments, theinvention is directed to an antibody formulation comprising an antibodywherein the antibody comprises a heavy chain variable region and a lightchain variable region, wherein the heavy chain variable region comprisesthe Kabat-defined CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 5-7, andwherein the light chain variable region comprises the Kabat-definedCDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 8-10, wherein the antibodyformulation is essentially free of particles for at least 1 month, atleast 2 months, at least 3 months, at least 4 months, at least 5 months,at least 6 months, at least 8 months, at least 10 months, at least 12months, at least 18 months, at least 24 months, at least 30 months, atleast 36 months, or at least 48 months when stored at 2-6° C.

In some embodiments, the antibody formulation is essentially free ofGST. In some embodiments, the term “essentially free of GST” refers toan antibody formulation having a GST activity of less than about 0.5units/mg antibody, less than about 0.3 units/mg antibody, less thanabout 0.1 units/mg antibody, less than about 0.08 units/mg antibody,less than about 0.05 units/mg antibody, less than about 0.03 units/mgantibody, less than about 0.01 units/mg antibody, less than about 0.005units/mg antibody, less than about 0.001 units/mg antibody, less thanabout 5×10⁻³ units/mg antibody, less than about 1×10⁻⁴ units/mgantibody, less than about 1×10⁻⁵ units/mg antibody, or less than about 11×10⁻⁶ units/mg antibody. In some embodiments, the term “essentiallyfree” refers to a level of GST that is non-detectable using common GSTdetection techniques.

Various methods to determined GST activity are known to those in theart. In some embodiments, the GST activity is determined using aGlutathione (GSH/GSSG/Total) Fluorometric Assay Kit (BioVision, SanFrancisco Calif.).

In some embodiments, the invention is directed to a method of purifyingan antibody comprising a heavy chain variable region and a light chainvariable region, wherein the heavy chain variable region comprises theKabat-defined CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 5-7, andwherein the light chain variable region comprises the Kabat-definedCDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 8-10, the methodcomprising (i) obtaining a cell culture comprising the antibody, (ii)performing affinity chromatography on the antibody, (iv) performingcation exchange on the antibody, (v) performing mixed modechromatography on the antibody. In some embodiments, the invention isdirected to a method of purifying an antibody wherein the antibodycomprises a heavy chain variable region and a light chain variableregion, wherein the heavy chain variable region comprises theKabat-defined CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 5-7, andwherein the light chain variable region comprises the Kabat-definedCDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 8-10, the methodcomprising (i) obtaining a cell culture comprising the antibody, (ii)binding the antibody to a Protein A column, (iii) eluting the antibodyfrom the Protein A column, (iv) performing cation exchange on theantibody, (v) performing mixed mode chromatography on the antibody. Insome embodiments, the method of purifying an antibody further comprisesa viral inactivation process. In some embodiments, the viralinactivation step is performed by lowering the pH to less than 4.0. Insome embodiments, the method further comprises a diafiltration process.In some embodiments, the method further comprises a filtration process.In some embodiments, the filtration process is sufficient to removeactive virus particles.

In some embodiments, the invention is directed to a method of treating apatient. In some embodiments, the method comprises administering theantibody formulations described herein, the containers described herein,the unit dosage forms described herein, or the pre-filled syringedescribed herein to a subject in need thereof.

In some embodiments, the invention is suitable for treatment ofpulmonary disease or disorder by administering the antibody formulationdescribed herein. In some embodiments, the invention is directed to amethod of treating a patient with an eosinophilic disease or disorder byadministering the antibody formulation described herein. In someembodiments, the invention is directed to a method of treating apulmonary disease or disorder in a subject, the method comprisingadministering the antibody formulations described herein. In someembodiments, the invention is directed to a method of treating aneosinophilic disease or disorder in a subject, the method comprisingadministering the antibody formulations described herein. In someembodiments, the invention is directed to treatment of pulmonarydiseases or disorders e.g., asthma, COPD, eosinophilic asthma, combinedeosinophilic and neutrophilic asthma, aspirin sensitive asthma, allergicbronchopulmonary aspergillosis, acute and chronic eosinophilicbronchitis, acute and chronic eosinophilic pneumonia, Churg-Strausssyndrome, hypereosinophilic syndrome, drug, irritant andradiation-induced pulmonary eosinophilia, infection-induced pulmonaryeosinophilia (fungi, tuberculosis, parasites), autoimmune-relatedpulmonary eosinophilia, eosinophilic esophagitis, or Crohn's disease orcombination thereof in a subject, the method comprising administeringthe antibody formulations described herein. In some embodiments, theinvention is directed to treatment of asthma in a subject, the methodcomprising administering the antibody formulations described herein. Insome embodiments, the invention is directed to treatment of COPD in asubject, the method comprising administering the antibody formulationsdescribed herein.

In some embodiments, a therapeutically effective amount of the antibodyformulations described herein is administered to treat a condition. Asused herein, the term “therapeutically effective amount” refers to theamount of a therapy (e.g., an antibody that immunospecifically binds toan IL-5 receptor polypeptide), that is sufficient to reduce the severityof a disease or disorder (e.g., a disease or disorder characterized byaberrant expression and/or activity of an IL-5 polypeptide, a disease ordisorder characterized by aberrant expression and/or activity of an IL-5receptor or one or more subunits thereof, an autoimmune disease, aninflammatory disease, a proliferative disease, or an infection(preferably, a respiratory infection) or one or more symptoms thereof),reduce the duration of a respiratory condition, ameliorate one or moresymptoms of such a disease or disorder, prevent the advancement of sucha disease or disorder, cause regression of such a disease or disorder,or enhance or improve the therapeutic effect(s) of another therapy. Insome embodiments, the therapeutically effective amount cannot bespecified in advance and can be determined by a caregiver, for example,by a physician or other healthcare provider, using various means, forexample, dose titration. Appropriate therapeutically effective amountscan also be determined by routine experimentation using, for example,animal models.

The terms “therapies” and “therapy” can refer to any protocol(s),method(s), and/or agent(s) that can be used in the prevention,treatment, management, or amelioration of a disease or disorder (e.g., adisease or disorder characterized by aberrant expression and/or activityof an IL-5 polypeptide, a disease or disorder characterized by aberrantexpression and/or activity of an IL-5 receptor or one or more subunitsthereof, an autoimmune disease, an inflammatory disease, a proliferativedisease, or an infection (preferably, a respiratory infection) or one ormore symptoms thereof). In certain embodiments, the terms “therapy” and“therapy” refer to biological therapy, supportive therapy, and/or othertherapies useful in treatment, management, prevention, or ameliorationof such a disease or disorder or one or more symptoms known to skilledmedical personnel.

As used herein, the term “therapeutic protocol” refers to a regimen fordosing and timing the administration of one or more therapies (e.g.,therapeutic agents) that has a therapeutic effective.

The route of administration of the antibody formulation of the presentinvention can be via, for example, oral, parenteral, inhalation ortopical modes of administration. The term parenteral as used hereinincludes, e.g., intravenous, intraarterial, intraperitoneal,intramuscular, subcutaneous, rectal or vaginal administration. In someembodiments, the antibody is an anti-IL5R antibody and the route ofadministration is intramuscular injection. While all these forms ofadministration are clearly contemplated as being within the scope of theinvention, in some embodiments, the antibody formulation is suitable foradministration via injection, in particular for intravenous orintraarterial injection or drip.

In some embodiments, the compositions and methods of the presentinvention enable a manufacturer to produce an antibody formulationsuitable for administration to a human in a more efficient manner,either by reducing costs, reducing method steps, reducing opportunitiesfor error, reducing opportunities for introduction of unsafe or improperadditives, reducing waste, increasing storage time, etc.

EXAMPLES Example 1

Formulation studies were performed to develop a stable anti-IL5Rantibody formulation that is appropriate for delivery of a dose between2-100 mg via subcutaneous delivery from pre-filled syringe (or a vialback-up configuration). Specifically, two separate antibodyconcentration formulations were developed, a 2-20 mg/mL formulation, anda 20-100 mg/mL formulation.

1. Materials and Methods

a. Source of Anti-IL5R and Preparation of Formulations

Multiple lots of anti-IL5R were used in these studies. All of the lotswere produced at various scales by MedImmune and delivered afterdiafiltration and concentration to approximately 130 g/L in 20 mMhistidine/histidine HCl at pH 6. Some lots also had 250 mM trehalose inthe diafiltration buffer.

Anti-IL5R was formulated by spiking in excipient addition buffer (EAB)to achieve an anti-IL5R concentration of 100 g/L and appropriateconcentrations of buffer and excipient species. Lower concentration drugsubstances were made from the 100 g/L formulation.

2. Accelerated Stress Methods

a. Storage at Increased Temperature

Vials and syringes were stored in controlled stability chambers tomaintain constant temperature during storage. The chambers weremaintained at 2-8° C., 23-27° C./60% RH, or 38-42° C./75% RH, but willbe referred to by their midpoint temperatures of 5°, 25°, or 40° C.henceforth. Vials were stored upright unless otherwise noted andpre-filled syringes were stored tip down.

b. Freeze-Thaw Cycles

Controlled and uncontrolled freeze-thaw cycles were both used in thesestudies. Uncontrolled freeze-thaw was done by freezing vials in a −40°C. chamber and thawing them at room temperature.

c. Transportation and Shaking

A variety of methods were used to investigate the effect oftransportation on anti-IL5R. Benchtop shaking of vials was done byorbital shaking at 150 rpm for 24 hours. Actual transportation wasmimicked by shipping the product to an off-site location. The productmade two round trips and undergoes ground and air transportation over 4days. A combination of frozen and cold packs were used to maintain theproduct temperature at 2-8° C., and was monitored by sensors thatindicate temperatures below 0° C. or above 9° C.

For screening studies, transportation was simulated using a vibrationtable (transportation simulator). The product underwent “air” and“ground” transportation in a similar pattern to that experienced duringround trip shipping. The process took 12 hours and again the temperaturewas controlled with cold packs to be 2-8° C.; sensors were not used.Horizontal orientation was chosen as worst-case orientation during realor simulated shipping due to the potential for bubble travel andpotential drug contact with the entire barrel, needle tip and stopper.

3. Experimental Methods

a. Methods Following or Deriving from an SOP

Visual inspections were performed by comparing to particle andopalescence standards. Aggregation and fragmentation were monitored bySE-HPLC. For anti-IL5R concentrations below 10 g/L, a larger volumeinjection was used to achieve similar total protein mass per injection.Some samples were also used for cIEF measurements and RP-HPLC to monitorfragmentation.

b. Protein Concentration

Protein concentration was measured by diluting the protein by serialgravimetric dilution to approximately 0.5 g/L and measuring theabsorbance at 280 nm. The concentration was calculated from theextinction coefficient and the dilution factor, and corrected for theeffect of density on gravimetric dilution for initial concentrationsabove 50 g/L.

c. Sub-Visible Particle Counts

Sub-visible particle counts were made using both the MFI and HIAC. Forthe MFI, 0.9 mL of solution was run neat after the optical illuminationwas run with water. The first 0.2 mL was used to purge the system andnot included in the analysis. An aspect ratio filter of <0.85 was usedto remove spherical air bubbles or silicone oil droplets. For HIAC,solutions with concentrations>5 g/L were diluted to approximately 5 g/L,while dilute samples were run neat. The dilution was done inside alaminar flow hood with 20 mM histidine/histidine HCl, pH 6 buffer thatwas filtered just before use. Samples were degassed under vacuum for atleast 30 minutes before testing. The average of three runs wasmultiplied by the dilution factor for the final result. Silicone oildroplets were not differentiated from protein particles by the HIAC.

4. Data and Discussion

a. Polysorbate-20 Concentration Screen

The first goal was to optimize the PS-20 concentration in the aqueousantibody formulation. Polysorbate was included in solutions to protectthe protein from denaturing and aggregating at interfaces, and therequired concentration was expected to be different in the liquidproduct than the lyophilized one. The major interfacial stresses wereencountered during freeze-thaw and transportation, so the experimentalplan focused on mimicking these. Prior experience indicated that 0.02%polysorbate 20 was sufficient to completely protect against both ofthese stresses (data not shown). As verification, these stresses werecombined in series, in the order expected for clinical production, andan incubation period was added post-stress to enable growth of anypotential particles. First, the drug substance underwent threeuncontrolled freeze-thaw cycles and the material was filtered and filledinto vials. Then it was shaken on the benchtop and incubated for oneweek at 40° C. before testing by SE-HPLC and MFI. The conditions testedwere the extremes of the concentration range, 2 and 100 g/L, formulatedin 240 mM trehalose, 20 mM histidine/histidine HCl, pH 6 with varyinglevels of PS-20 from 0-0.03% w/v. The results are found in FIGS. 2, 3,and 4.

The monomer fraction data indicated that 2 g/L solution remained pureregardless of the polysorbate level, but that low amounts ofpolysorbate-20, below approximately 0.005%, caused a small amount ofaggregation at 100 g/L, but these results did not in themselves indicatean “edge of failure.” The stresses utilized were severe, and thedegradation level minimal, thus any level of PS-20 tested could beadequate from an SEC aggregate perspective. At 2 g/L, the sub-visibleparticle counts were quite high and were not controlled bypolysorbate-20 in the range tested. At 100 g/L, high sub-visibleparticle counts were controlled by the presence of 0.003% or more PS-20.Together this data indicated PS-20 levels should be maintained at orabove 0.003%.

An alternative method of controlling sub-visible particles was requiredfor low concentration solutions, as is discussed below.

b. Screen of the pH

The effect of solution pH was studied in 2, 20, and 100 g/L solutionswith pH values of 5 to 7.5. The rest of the formulation was constant andincluded 240 mM trehalose, 20 mM histidine/histidine-HCl, and 0.02%PS-20. Solutions were prepared and stored at 40° C. for one month beforetesting. Monomer loss by SE-HPLC, subvisible particles by MFI, andvisible particles by visual inspection were evaluated for all samples.Additional testing was run on the 100 g/L samples, including RP-HPLC andcIEF. Results are provided in FIGS. 5 and 6.

Aggregation and fragmentation were minimized within the pH range of5.5-6.5. The results from cIEF were inconsistent with the referencestandard at pH 7.0 and above. Sub-visible particle counts were eitherlow or did not show any pattern with solution pH. Visible particlescores were higher from pH 5.5-6.5. Though these scores are high, thesamples were inspected close to the light where higher particle countsare routinely seen. It was possible that the source material alsocontributed to high particle levels, as this was material that had HCPlevels reduced by further purification over protein-A. This studyindicated that an optimal pH from a particle formation perspective wouldbe pH 5 or pH>7. However, since the visible particle scores wereunderstood to be overestimates here, the pH was not be changed from pH6.0 as a result of this study.

c. Effect of Shipping

The formulations needed to be robust to shipping, so they were tested atvarious protein and PS-20 concentrations in pre-filled syringes. Theformulation varied from 2-100 g/L anti-IL5R and 0-0.05% PS-20, withother conditions constant at 240 mM trehalose, 20 mM histidine/histidineHCl, pH 6. A number of other conditions were tested at 2 g/L, includingglycine, calcium chloride, pH 5.5, pH 6.5, and 0.02% polysorbate-80.None of these conditions showed improvement over the trehaloseformulation at pH 6 with polysorbate-20, so they are not discussedfurther.

One mL of sample was filled into a platform PFS with 0.4 mg of siliconeoil. The samples were shipped, stored at 5° C., 25° C., and 40° C. andtested over two months by visual inspection, MFI, and HIAC. Results arepresented in FIG. 7.

The plot shows sub-visible particle counts from MFI after 1 month ofstorage at 25° C. Sub-visible particle counts from HIAC or after storageat other temperatures showed similar trends to the data set shown.Visual inspections did not indicate high visible particle counts for anyof the samples except the one containing calcium chloride. The highprotein concentration solutions (>20 g/L) were robust to shipping aslong as some PS-20 is present. Therefore, the trehalose formulation wasused in long term stability studies for 20-100 g/L solution.

The shipping data verified that the low concentration formulations werenot robust, as observed by the high and highly variable sub-visibleparticle counts. The data also showed that the issue was not solved bypolysorbate alone, therefore the low concentration solutions needed tobe reformulated.

d. Reformulation of Low Concentration DS

The low concentration reformulation screens were stressed by simulatedtransportation and tested by MFI. The sub-visible particle counts shownwere particles>10 μm, aspect ratio filtered; trends were similar forother particle sizes.

During manufacturing, an unformulated drug substance (UDS) at highconcentration (>100 g/L) containing trehalose will be produced andfrozen. Storage of this high concentration intermediate is necessary toenable dilution into various formulations across the 2-100 mg/mLformulation range. Dilution from the UDS will result in some residualtrehalose; for uniformity of composition, a single trehaloseconcentration will be used for the entire low dose range. The buffer andpH were not changed.

The first screen was used to determine the minimum protein concentrationwhere the trehalose formulation was stable, and to determine the effectof increased ionic strength by formulating in 150 mM trehalose with 75mM arginine HCl or sodium chloride. The results shown below indicatethat protein concentrations>10 g/L were stable, but for robustness thelow concentration range was set at 2-20 g/L. Increasing the ionicstrength resulted in more stable solutions with both excipients. See,e.g., FIG. 8.

The next studies focused on optimizing the arginine or NaClconcentrations; a protein concentration of 2 g/L was used as worst casefor all the studies. For each excipient, broad and narrow excipientconcentration screens were run with 0.02% PS-20. The initial argininescreen was run with varying amounts of trehalose, where solutions weremade by combining 270 mM arginine with 250 mM trehalose. The objectivewas to maintain the osmolality, but the calculation was done incorrectly(arginine-HCl is bivalent) so the arginine containing solutions werehyper-osmotic. The rest of the excipient concentration screens were donewith a constant trehalose concentration of 40 or 50 mM, based on theresidual trehalose at 20 g/L after dilution from the 100 g/L stock. Theresults are provided in FIGS. 9 and 10.

The results of the broad arginine and NaCl screens indicated that theconcentration needs to be greater than 50 mM arginine or 75 mM NaCl toproduce a stable formulation. The narrow concentration screens of 75-150mM arginine or 100-200 mM NaCl resulted in low particle countsthroughout the entire range. This indicated a concentration in themiddle of these ranges should yield a robust formulation; 130 mM waschosen to be iso-osmotic in combination with 50 mM residual trehalose.

The PS-20 concentration optimization was checked for the newformulations using the same method. These experiments were doneconcurrently with the narrow excipient concentration screens, so amid-point concentration was used. The conditions tested were 0.01-0.1%PS-20, 115 mM arginine HCl, 40 mM trehalose and 0.01-0.05% PS-20, 150 mMNaCl, 50 mM trehalose. A couple of samples were tested with 0.02% PS-80,but higher counts resulted than the corresponding PS-20 result (data notshown). The particle counts were low for all of the polysorbate levelstested, indicating that the level does not need to be changed from 0.02%in the new formulations. See, e.g., FIG. 11.

The results of the reformulation for the low concentrations of anti-IL5Rindicated either arginine or NaCl were able to stabilize the formulationin the short term. The formulations considered were 130 mM arginine HClor 130 mM NaCl, with 50 mM trehalose, 20 mM histidine/histidine HCl,0.02% PS-20, pH 6 for protein concentrations from 2-20 g/L.

e. Vial and PFS Considerations

The three formulations developed above (20-100 mg/mL in trehalose, 2-20mg/mL in trehalose/arginine, and 2-20 mg/mL in trehalose/NaCl) wereappropriate for both vial and PFS configurations. The vial configurationwas a 3 cc Schott vial with a 4432/50 West stopper. The highest riskwith regard to the vial configuration was the silicone oil level on thestoppers, so the long term stability studies were run with stoppers thathave a higher level of silicone oil (0.039 mg/cm²) than those that willbe generally be used (0.007-0.024 mg/cm²).

The syringe tested with the anti-IL5R formulation was the platformsyringe, a BD 1 mL long PFS with a clipped flange, a staked 29 G thinwall needle, containing 0.4 mg Si oil, and covered with a BD260 rigidneedle shield (Catalog #47363119).

f. Long Term Stability Studies

Two long-term stability studies were run to verify the decisions madefrom screening studies. Stability study #1 investigated the long termstability of the trehalose and arginine/trehalose formulations in PFSand vials. Additionally, PFS comparisons were made which will not bediscussed here. Stability study #2 was initiated to provide data fromanother lot of material for the configurations examined in study #1, andalso to investigate the NaCl/trehalose formulation bracket and theimpact of fill volume from ½ mL to 1 mL in PFS and vials.

i. Stability Study #1: Stability of Anti-IL5R PFS Presentations

Stability studies were run in syringes with vials as a control. Each ofthe end points of the formulation brackets was tested in each primarycontainer. The syringe tested was the platform Hypak™ for Biotech; thisis a BD glass 1 mL long syringe, practically free of tungsten with a 29G thin wall needle and 0.4 mg of silicone oil. The vials used were 3 ccSchott vials with West 4423/50 stoppers and over-seals.

The formulations filled are the following:

-   -   2 and 20 mg/mL anti-IL5R antibody, 125 mM arginine HCl, 50 mM        trehalose, 20 mM histidine/histidine HCl, 0.02% PS-20, pH 6; and    -   20 and 100 mg/mL anti-IL5R antibody, 250 mM trehalose, 20 mM        histidine/histidine HCl, 0.02% PS-20, pH 6.

A. Purity of Anti-IL5R Pre-Filled Syringe Presentations

There was no significant effect of the primary container on monomer lossfor any formulations. Some impact of protein concentration was observed,but the monomer loss rate was consistently low. See FIG. 12A.

B. Particle Analyses of Anti-IL5R Pre-Filled Syringe Presentations

Particle formation was thought to be the main degradation route foranti-IL5R so it was thought to play a large role in determining asuitable PFS. Sub-visible particle measurements by HIAC showed anincrease in the number of particles in the PFS, likely due to siliconeoil droplets (See, FIGS. 12B and 12C). However, the total particlecounts remain well below the USP limits of 6000 particles>10 μm/mL and600 particles>25 μm/mL for all configurations. MFI was used as anorthogonal method and showed similar results, though less of adifference was observed between containers since silicone oil dropletscan be filtered out of the results in the MFI software.

ii. Summary of Stability in Hypak™ for Biotech Syringes at 5° C.

TABLE 1 shows a summary of the stability data (Stability Study #1)available for anti-IL5R in the Hypak™ for Biotech syringes at 5° C. upto 16 months for arginine formulations and 24 months trehaloseformulation. Visual particles were detected which lead to a reduction inthe PS-20 concentration from 0.02% to 0.006%. No other high risks wereidentified, though sub-visible particle counts were variable.

TABLE 1 Range of results for all time points tested 2 mg/mL 20 mg/mL 20mg/mL 100 mg/mL Assay Arg Arg Tre Tre Appearance (Visible Particles,<STD 1 =STD 4 =STD 2 <STD 3 worst observation) HIAC >10 μm <590 <3,000<1,800 <2,000 (Particles/mL) >25 μm  <30 <20 <30   <60 MFI >10 μm <110<2,200 <1,400 <1,700 (Particles/mL) >25 μm  <50 <100 <60   <480* SEC(Mon. Loss %/yr)     0% 0.1% 0.1%      0.2% RP (% Fragment)  ≤2.0% ≤1.8%≤1.9%    ≤1.9% Functionality Forces by Instron <6N <7N <7N <12N(Break-loose and Glide Force) BioAssay (Potency %) 93-116% 92-103%95-111% 88-99% BioAnalyzer Reduced Consistent with Ref. Std. Non-ReducedConsistent with Ref. Std. cIEF Consistent with Ref. Std. Stabilitysummary of anti-IL5R in multiple formulations in Hypak for Biotechsyringes at 5° C. for 16 months. Appearance results included particles,which were mitigated by reducing the PS-20 concentration (covered in aseparate report). Sub-visible particle results were variable, but notrends were observed. All other results are within expectations for astable product.*

iii. Stability Study #2

Stability study #2 was a bracketed stability study in pre-filledsyringes and vials, designed to decide the low dose formulation and thefill volume, and to verify placebo stability.

The previous stability study used to choose the PFS had a fill volume of1 mL only. However, there were a number of potential benefits to a lowerfill volume including reduced pain on injection, a smaller lump underthe skin, faster administration, and less leakage from theadministration site.

The bracketing strategy for the 1 mL fill option was two brackets ofprotein concentration, 2-20 g/L for the low dose and 20-100 g/L for thehigh dose. The same brackets for a 12 mL fill covered the dose range of1-50 mg, and also required a fill volume bracket of 100 g/L from ½-1 mLfor the 50-100 mg dose. This is shown graphically in FIG. 13 forclarity. In both cases, the lowest dose bracket was studied in both thearginine and NaCl based formulations.

The drug substances and placebos filled were the following:

-   -   All solutions: 20 mM Histidine/Histidine HCl, 0.02% PS-20, pH        6.0    -   2 Arg/20 Arg: 2 or 20 g/L anti-IL5R, 125 mM Arginine HCl, 50 mM        Trehalose    -   2 NaCl/20 NaCl: 2 or 20 g/L anti-IL5R, 130 mM NaCl, 50 mM        Trehalose    -   20 Tre/100 Tre: 20 or 100 g/L anti-IL5R, 250 mM Trehalose    -   Arg Placebo: 125 mM Arginine HCl, 50 mM Trehalose    -   NaCl Placebo: 130 mM NaCl, 50 mM Trehalose    -   Tre Placebo: 250 mM Trehalose

The 1 mL fill volume configuration was not placed on test for any of thevial configurations or the placebo in PFS in order to reduce the totalsize of the study, since the ½ mL fill volume was the most likelyworst-case configuration due to the higher surface area to volume ratio.

All of the samples were shipped and then placed on stability at 5, 25,and 40° C. The vials were stored inverted during stability to maximizecontact with the stopper. At the time the visible particles werediscovered in older studies, 6 weeks of data had been collected for thisstability study. Additionally, particles were observed in the NaClformulation between the 3 and 6 month time points of this study (datanot shown), leading to the NaCl formulation being rejected in favor ofthe arginine formulation.

The additional formulation work required to mitigate the formation ofvisible particles is described in the examples that follow and resultedin a decrease of the polysorbate-20 concentration from 0.02% to 0.006%.No other stability concerns were observed for this formulation, and nosignificant impact of container or fill volume was observed. One year ofstability data is summarized in the FIG. 14, including visible particlescores, purity loss by SEC, particle counts by HIAC, and potency (notall configurations were tested at all time points).

Conclusion of Example 1

Formulation screening was carried out using various stress methodsincluding freeze/thaw, agitation, silicone oil spiking, and acceleratedstability. Long-term stability was used to verify the results of thescreening studies. The phase 2b trehalose formulation was successful forliquids at high concentration with an increased polysorbateconcentration, but showed instability when extended to liquids at lowconcentrations, primarily through the formation of sub-visibleparticles. The low concentration range was reformulated by increasingthe ionic strength with either arginine hydrochloride or sodiumchloride, resulting in a stable solution.

In order to cover the wide range of possible doses (2-100 mg), the threepotential formulation brackets were the following:

-   -   2-20 g/L, 130 mM arginine hydrochloride, 50 mM trehalose        dihydrate, 20 mM histidine/histidine hydrochloride, 0.02%        polysorbate-20, pH 6.0;    -   2-20 g/L, 130 mM sodium chloride, 50 mM trehalose dihydrate, 20        mM histidine/histidine hydrochloride, 0.02% polysorbate-20, pH        6.0; and    -   20-100 g/L, 250 mM trehalose dihydrate, 20 mM        histidine/histidine hydrochloride, 0.02% polysorbate-20, pH 6.0.

Long-term stability of up to 24 months indicates all three formulationswere stable at 2-8° C. with respect to agitation, relatively insensitiveto silicone oil, and compatible with vials and PFS. Additionally,minimal degradation was observed at elevated temperatures. Continuedobservations of the two low concentration formulations eliminated theNaCl option due to increased visible particle formation compared to thearginine option, resulting in two formulation brackets. Data indicatedpre-filled syringes are an acceptable primary container with either a 1mL or ½ mL fill volume. Visible particle formation remained a problemwith these formulations, as addressed in the following examples.

Example 2—Particle Formation in Anti-IL5R Formulations

Visible particles were detected in aqueous formulations of anti-IL5R inboth vials and PFS in previous long term stability studies, with thefirst incidence of detection at the 6 month time point (data not shown).A study was conducted to mitigate visible particle formation in ananti-IL5R antibody formulation comprising polysorbate 20 (PS-20) storedfor an extended period of time. The following example describes a longterm study of formulations comprising various PS-20 and proteinconcentrations to verify the importance PS-20 concentration has inmitigating particle formation. This study resulted in an acceptablerange of 0.002-0.01% PS-20.

Abbreviations and Definitions

Abbreviation Definition Abbreviation Definition cIEF CapillaryIsoelectirc MFI Micro-Flow Focusing Imaging DLS Dynamic Light PSPolysorbate Scattering DP Drug Product PFS Pre-filled syringe DS DrugSubstance RP Reverse Phase Chromatography FC Flow Cytometry SEC SizeExclusion Chromatography Abbreviation Definition Arg Arginineformulation: 130 mM arginine HCl, 50 mM trehalose dihydrate, 20 mMhistidine/histidine HCl, pH 6.0, various PS-20 Tre Trehaloseformulation: 250 mM trehalose dihydrate, 20 mM histidine/histidine HCl,pH 6.0, various PS-20

Preliminary Studies

Particle formation was detected in the long term stability studiesdescribed in Example 1. However, the particles were extremely small andappear more like a cloud than individual particles. In order to see theparticles, the samples were inspected close to the light source. Sincethe particles were dissimilar to the ones in the vial and PFS standardsets, the samples were compared to each other as well as the standardsat each time point.

Particles were first detected at the 6 month time point in 100 g/L Trevials with 0.02% PS-20.

Six months and 11 month old samples were compared in (1) vials and (2)pre-filled syringe (PFS). Both studies showed that particle formationwas more severe in vials than PFS, though PFS standards weren'tavailable at the time so a numerical comparison is not available.Samples from PFS were decanted and injected into vials for appearancetesting. No more particles were observed in these vials, which verifythat the difference is not a path length effect. In trehaloseformulations, this vial-to-PFS difference is more significant than inarginine formulations.

A comparison of various PS-20 concentrations (0, 0.01, 0.02 and 0.03%)in 100 g/L Tre formulations in both vials and PFS after shipping wasmade. The lower PS-20 concentrations of 0 and 0.01% PS-20 werecompletely free of particles at the 11 month time point. Particles werevisible in both the vial and PFS at 0.02% and 0.03% PS-20. At 20 months,the 0 and 0.01% PS-20 PFS and 0% PS-20 vial remained clear but the 0.01%vial had a very small tornado of particles.

In targeted studies, when held close to the light source, particles wereclearly visible in the 100 g/L Tre, 0.02% PS-20 vials and PFS, in somecases as early as 3-4 months. At the low concentration (20 g/L) end ofthe Tre bracket particles formed much more slowly and were only observedin vials at 21 months. No particles have been observed in 20 g/L Tre PFSat any PS-20 concentration, with data available up to 21 months.Particle formation in arginine formulations was somewhat slower, withparticles being observed in the high concentration (20 g/L) end of thebracket with 0.02% PS-20 at 6-9 months. Particles have never beenobserved in the 2 g/L Arg formulation at any PS-20 concentration ineither container, with data available up to 16 months.

Accelerated and stressed temperature studies did not provide insightinto particle formation. At 40° C., particles did not form at all overthe testing period of 3 months. At 25° C., particle formation wassimilar or slightly reduced compared to 5° C.

This data indicated the high protein concentration ends of the bracketwere at high risk for visible particles and the low ends werepotentially at risk in the long term. Reducing the PS-20 concentrationwas investigated to mitigate particle formation. The primary containerremained the platform PFS, and vials were used as an early read onparticle formation since particle formation in vials was more severe andeasier to see.

Investigation of Various PS-20 Concentrations

An extensive stability study was designed to investigate particleformation in various formulations, various PS-20 concentrations, andvarious antibody concentrations. All samples were filled into both PFSand vials, shipped twice to a separate location to simulate thedistribution process, and placed on stability at 5° C. Appearancetesting was performed monthly. SEC, HIAC, and MFI were also tested for asubset of samples at time zero, 3, 6, and 9 months. FIG. 15 shows thesamples that were prepared as part of this study.

Without any PS-20, sub-visible particles formed upon shipping as shownby the MFI data in FIG. 16 for PFS at time zero. The lowest tested levelof PS-20 (0.002%) or more was sufficient to protect from shippingstress. Additionally, the lowest PS-20 concentration (0.002%) wasverified to be sufficient to protect from DS shipping stress in a tankusing a scale down model (data not shown here).

The appearance results for particles showed that there was significantparticle formation in vials above 0.01% PS-20, and some particles in PFSat 0.02% PS-20 for both formulation brackets, with fewer particlesobserved at the lower concentration end of both brackets. See, FIG. 17.The first observations of the particles were at 3 months for 100 g/L Treand at 6 months for 20 g/L Arg. The figures illustrate the impact ofprotein and PS-20 concentration on particle formation at the latest timepoint, 9 months. It is noted that the particle formation observationswere all made close to the light source.

The appearance data sets an upper limit on the PS-20 concentration of0.01% and the sub-visible particle counts set a lower limit of 0.002%(from the available data, actual limit may be lower). The midpoint ofthis acceptable range (0.006%) was set as the new PS-20 concentration.

Overall stability was verified for PFS for the bracket end samplesincluding up to 9 months of data for 2 g/L Arg and 20 g/L Arg, and up to18 months of data for 20 g/L Tre and 100 g/L Tre. The following assayswere tested:

A. 0.002, 0.006, and 0.01% PS-20: Appearance, HIAC, MFI, and SEC.

B. 0.006% PS-20 only: Instron, BioAssay, BioAnalyzer, RP, and cIEF.

All of the data indicates the new formulation was stable and low risk. Asummary of the data is shown in Table 2.

TABLE 2 Results for all Assay samples tested Appearance (VisibleParticles, <Std 2 worst observation) HIAC >10 μm <640/mL(Particles/mL) >25 μm  <30/mL MFI >10 μm <610/mL (Particles/mL) >25 μm <60/mL SEC (Monomer Loss %/yr) 0.0-0.4%/yr    RP (% Fragment) ≤1.8%Functionality Forces by Break-loose Force  ≤8.5N Instron Glide Force≤11.8N BioAssay (Potency %) 86-109% BioAnalyzer Reduced Consistent withRef. Std. Non-Reduced Consistent with Ref. Std. cIEF Consistent withRef. Std.

The available data from Example 2 indicated a reduction of the PS-20concentration to 0.006% mitigates particle formation and produced anantibody formulation product that was stable for at least 9 months(arginine formulation) or 18 months (trehalose formulation), with noindications of an upcoming failure.

Example 3 Orthogonal Methods for Particle Detection

The primary method of particle detection and quantification wasappearance testing by visual inspection as exemplified in Example 2.Visual inspection was variable for a number of reasons. In general,visual inspection varied due to the innate variability of humanperception, leading to different results for different individuals. Dueto the very small size of these particles their visibility was highlydependent on the amount of light and they were dissimilar to theparticle standards for both vials and PFS. These factors increased thevariability of results between time points and between analysts.

Orthogonal methods were investigated to verify the appearance results.The particles formed in trehalose formulations were too small to seeindividually, so sub-visible particle methods were investigated. Theworst case sample (100 g/L, Tre, 0.02% PS-20, vial) from various lots at2 weeks, and 2, 5, and 9 months old was compared by DLS, FC, HIAC, andMFI.

The DLS was run at 100 g/L, leading to an underestimate of the main peakas expected and unreliability of the all peak sizes. Large peaks weredetected for the 2, 5, and 9 month old samples that were 1.4-2.2 μm insize. Although the reported size was not reliable, the presence of thepeaks correlated with visible particles. However, neither the reportedsize of the particles nor the intensity of the particle peak correlatedwith the visual appearance results.

HIAC results were similar for all of the samples; the particles were notdetected.

MFI counts for particles>10 μm and >25 μm were similar for all samples.However, MFI counts for particles>1 μm and >2 μm and FC counts trendwith the visual appearance results and increase as a function of thesample age. The results for these samples are shown in FIG. 18.

Further experiments indicate that particles>1 μm by MFI provide morereliable trends with visual appearance results than larger particles orFC counts (data not shown).

Similar experiments were run with 20 g/L Arg samples showing visibleparticles, but none of the orthogonal methods were successful fordetecting these particles. The particles formed in arginine formulationsappeared much larger than those formed in trehalose and were seen asindividual particles, which was likely why they were not detected bysub-visible particle methods.

MFI was used to verify the effect of PS-20 concentration in Example 2.The comparison of MFI and the appearance score are shown in FIG. 19 forExample 2 at the 9 month time point. Along with additional measurements(not shown), the comparison indicated particles were visible when theMFI count exceeded approximately 100,000 particles>1 μm/mL. The MFIresults provided added support to the conclusion that particle formationwas mitigated by 0.002-0.01% PS-20, especially in PFS. The vial data wasconsidered worst case and was also stable at the target concentration of0.006% PS-20.

Example 4 Stability Study

An additional stability study was performed to investigate all theconfigurations of the previous studies with the new target PS-20concentration of 0.006%, and to add confidence that these formulationsare stable. The bracket included a protein concentration bracket as wellas a fill volume bracket and is shown graphically in FIG. 20.

The addition of the fill volume bracket increased the range of dosesthat were covered by the trehalose formulation. These additionalconfigurations reduced the risk associated with the arginineformulation, which was riskier because the particles form more slowlyand cannot be detected by orthogonal methods. The samples included inthis stability study were:

-   -   0, 2, and 20 g/L, 1 mL fill, Arginine formulation, 0.006% PS-20;    -   0 and 2 g/L, 0.3 mL fill, Arginine formulation, 0.006% PS-20;    -   0, 20, 50, and 100 g/L, 1 mL fill, Trehalose formulation, 0.006%        PS-20; and    -   0 and 20 g/L, 0.3 mL fill, Trehalose formulation, 0.006% PS-20.

The samples were shipped to an off-site location twice to simulate thedistribution process and then placed on stability at 5° C., 25° C., and40° C.

Nine months of data was collected for the arginine formulations andtwelve months of data has been collected for the trehalose formulations.The results were consistent with historical data and no particles wereobserved in these samples up to this point. Further, no trends insub-visible particles over time have been observed, although a fewmoderately high outliers have occurred. The range of results for all ofthe assays is shown in Table 3.

TABLE 3 Range of results for all samples tested 5° C. (9 mos. Arg or 12mos. Tre Assay formulations) 25° C. (6 mos.) 40° C. (1 mo.) Appearance(Visible =Std 0 <Std 1 =Std 0 Particles, worst observation) HIAC >10 μm<2,400/mL   <1,900/mL <1,900/mL   (Particles/mL) >25 μm <110/mL  <110^(†)/mL <100/mL  MFI >10 μm <100/mL    <700/mL <50/mL(Particles/mL) >25 μm  <50/mL    <40/mL <10/mL SEC (Monomer Loss)0-0.3%/yr    0.1-0.6%/mo 2.2-3.5%/mo    RP (% Fragment) ≤1.7% ≤3.6%≤4.4% Functionality Forces by ≤11.7N Not tested Not tested Instron(Break-loose and Glide force) BioAssay (Potency %) 84-122% 83-113%83-110% BioAnalyzer Reduced Consistent with Ref. Std. Non-ReducedConsistent with Ref. Std. cIEF Consistent with Ref. Std. Inconsistentwith Ref Std. at 1 mo. ^(†)43 of 44 measurements fell in this range, butone outlier of 347 particles/mL was also measured.

The data supported the recommendation of this formulation bracket forthe full dose range. The trehalose bracket was lower risk than thearginine bracket and was recommended for doses as low as 6 mg.

Conclusion of Example 2-4

Particle observations in previous long-term stability studies led to aninvestigation into formulation variables that could be altered toimprove stability. The key variables, protein and polysorbateconcentration, were tested and analyzed. Based on the data presentedabove, an allowable range of polysorbate from 0.002% to 0.01% and atarget of 0.006% was determined to be optimal formulations for theanti-IL5R antibody formulation. This observation was supported by twostability studies with 18 and 12 months of available data, primarily byvisual appearance testing.

It was also observed that the trehalose formulation could be used ratherthan the arginine formulation in the 6-20 mg range by using a 0.3-1.0 mLvolume bracket at 20 mg/mL. The trehalose formulation was found to bemore predictable than the arginine formulation due to the larger dataset and better detection.

Example 5

Additional purification development was undertaken focused on reducinghost cell protein (HCP) to determine the effect of HCPs on particleformation. Anti-IL5R was purified as outlined in FIG. 21

2D gel analysis of the flow-thru of the Protein A column indicated therewere several protein species present. See, e.g., FIG. 22. The majorimpurities, as determined by reverse phase mass spectroscopy (RP-MS)included about 60% Fab fragment, about 5% light chain (LC) andfragments, and about 40% non-anti-IL5R related host cell proteins (HCP).The non-anti-IL5R related HCPs included glutathione S-transferase (GST),fructose-biphosphate aldolase, and transaldolase.

The flow-thru of the Protein A column was further passed through aProtein L column to further separate (1) the Fab fragments from (2) thenon-anti-IL5R HCPs. It was found that some material in the non-anti-IL5RHCPs was responsible for particle formation, and not the Fab fragments(data not shown).

To determine which of the major HCPs was responsible for particleformation, GST was first investigated. The role of GST in particleformation was investigated by (1) selective removal of GST to determineeffect on particle formation, (2) analysis of particle pellets todetermine the presence of GST in the pellet, and (3) adding GST toanti-IL5R formulations to determine effect on particle formation.

Preliminary evidence of specific removal of GST using an affinity matrixmade from a glutathione-sepharose conjugate suggested that removal ofGST resulted in reduced particle formation (data not shown). Analysis ofpellets formed from Protein A flow-thru indicated high concentrations ofGST in the pellets (data not shown).

GST was added (i.e., spiked) to purified anti-IL5R formulations lackingparticles to determine the effect of GST on particle formulations. GSTwas obtained from a commercial source (Prospec). GST was added to aformulation comprising 50 mg/mL anti-IL5R, 20 mM histidine-HCl buffer,9% (w/v) trehalose, 0.02% PS-20, pH 6.0, resulting in GST concentrationsof 3.8 μg/mg and 7.6 μg/mg. The samples were incubated at 38-42° C.Particles were observed by placing the samples on a light box. Theincubation time required to observe particle formation was dependent onthe level of GST present. For both the 3.8 rig/mg and 7.6 rig/mg spikedGST samples, particles were observed after 24 hours at 38-42° C. (datanot shown).

These results suggested that spiking GST into anti-IL5R formulationscauses particle formation in the anti-IL5R formulation.

Example 6

GST activity in various anti-IL5R formulations purified by various meanswas investigated. As a preliminary matter, an activity assay for GST(BioVision) was used to determine GST concentrations to form a standardcurve. GST catalyzes the formation of the thiol group of glutathione(GSH) to electrophilic compounds such as 1-chloro-2,4-dinitrobenzene(CDNB) for form a GS-DNB conjugate which is detected at 340 nM. Thus,the increased in absorption at 340 nM is directly proportional to theGST activity. Using this assay, the concentration of GST was determinedfor various anti-IL5R formulations (Samples A-J) purified using variousprocedures. The correlation between GST concentration and particleformation was noted. The results are provided in Table 4.

TABLE 4 Sample GST concentration Particle Description A 188.4 Highnumber of particles (the most) B 0.422 Practically free* C 2.074Practically free* D 1930.5 High number of particles E 1950.6 High numberof particles F 1774.2 High number of particles G 40.6 Particle forming H1.073 Some particles I <LLOQ Free of particles J 5.416 Particle forming*Some, but not many, particles. LLOQ = lower limit of quantification.

This evidence confirms that the presence of GST correlates with theformation of particles.

Example 7

Various purification columns were investigated to identify the mostefficient method of reducing the GST concentration in an anti-IL5Rformulation. See Table 5. GST concentration was determined as outlinedin Example 7.

TABLE 5 Description GST Conc. (μg/ml) CM Product 5.935 HA product 1.255MabSelect Sure Elut. Product <LLOQ CaptoAdhere Product <LLOQ CEXProduct* 9.228 CEX Product* 21.081  *GST concentrations of two separatebatches of anti-IL5R antibodies were determined.

Table 5 suggests that both the Protein A column (MabSelect Sure) and themixed mode chromatography column (CaptoAdhere) were successful atreducing the concentration of GST below a detectable level duringpurification of the anti-IL5R antibody.

Example 8

The presence of GST in other antibody preparations was investigated. Thepresence of particles in those antibody preparations was alsoinvestigated. The results are presented in Table 6, with comments.

TABLE 6 Trend with Antibody Active GST Particle formulation Detected?Formation? Comments Anti-IL5R Yes Yes N/A A Yes N/A Insufficientparticle data to determine trend B No (except 1 No Suggests that GST maynot be sample) the cause of particle formation for this Ab. C Yes N/AActive GST, but no particle formation D Yes N/A Insufficient Particledata to determine trend

All of the various embodiments or options described herein can becombined in any and all variations. While the invention has beenparticularly shown and described with reference to some embodimentsthereof, it will be understood by those skilled in the art that theyhave been presented by way of example only, and not limitation, andvarious changes in form and details can be made therein withoutdeparting from the spirit and scope of the invention. Thus, the breadthand scope of the present invention should not be limited by any of theabove described exemplary embodiments, but should be defined only inaccordance with the following claims and their equivalents.

All documents cited herein, including journal articles or abstracts,published or corresponding U.S. or foreign patent applications, issuedor foreign patents, or any other documents, are each entirelyincorporated by reference herein, including all data, tables, figures,and text presented in the cited documents.

1.-23. (canceled)
 24. A stable, aqueous antibody formulation comprising:a. about 2 mg/mL to about 100 mg/mL of an antibody, wherein the antibodycomprises a heavy chain variable region and a light chain variableregion, wherein the heavy chain variable region comprises theKabat-defined CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 5-7, andwherein the light chain variable region comprises the Kabat-definedCDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 8-10, and b. about 0.002%to about 0.01% polysorbate-20.
 25. The antibody formulation of claim 24,further comprising an uncharged excipient.
 26. The antibody formulationof claim 25, wherein the uncharged excipient is trehalose.
 27. Theantibody formulation of claim 24, comprising about 20 to about 100 mg/mlof the antibody.
 28. The antibody formulation of claim 25, wherein theuncharged excipient concentration is about 200 mM to about 400 mM. 29.The antibody formulation of claim 24, further comprising histidine. 30.The antibody formulation of claim 29, wherein the histidineconcentration is about 15 mM to about 30 mM.
 31. The antibodyformulation of claim 24, wherein said antibody was not subjected tolyophilization.
 32. A stable, aqueous antibody formulation comprising:a. about 30 mg/mL of an antibody, wherein the antibody comprises a heavychain variable region and a light chain variable region, wherein theheavy chain variable region comprises the Kabat-defined CDR1, CDR2, andCDR3 sequences of SEQ ID NOs: 5-7, and wherein the light chain variableregion comprises the Kabat-defined CDR1, CDR2, and CDR3 sequences of SEQID NOs: 8-10, b. about 0.006% polysorbate-20, c. about 250 mM trehalose,and d. about 20 mM L-histidine.
 33. The antibody formulation of claim24, wherein the formulation is stable upon storage at about 25° C. forat least 3 months.
 34. The antibody formulation claim 24, wherein theformulation is substantially free from particles upon storage at about40° C. for at least 1 month as determined by visual inspection.
 35. Theantibody formulation of claim 25, wherein the formulation is aninjectable formulation.
 36. A sealed container containing the antibodyformulation of claim
 24. 37. A pharmaceutical unit dosage form suitablefor parenteral administration to a human which comprises the antibodyformulation claim 24 in a suitable container.
 38. The pharmaceuticalunit dosage form of claim 37, wherein the suitable container is apre-filled syringe.
 39. The pharmaceutical unit dosage form of claim 38,wherein the pre-filled syringe comprises a needle.
 40. Thepharmaceutical unit dosage form of claim 39, wherein the needle is a 29G thin wall needle.
 41. The pharmaceutical unit dosage form of claim 38,wherein the pre-filled syringe is coated with silicone.
 42. A prefilledsyringe comprising: a. about 30 mg/mL of an antibody, wherein theantibody comprises a heavy chain variable region and a light chainvariable region, wherein the heavy chain variable region comprises theKabat-defined CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 5-7, andwherein the light chain variable region comprises the Kabat-definedCDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 8-10, and b. about 0.006%polysorbate-20.
 43. The pre-filled syringe of claim 43, furthercomprising c. about 250 mM trehalose.
 44. A kit comprising theformulation of claim
 24. 45. A method of producing a stable, aqueousantibody formulation, the method comprising: a. purifying an antibody toabout 1 mg/mL to about 400 mg/mL, wherein the antibody comprises a heavychain variable region and a light chain variable region, wherein theheavy chain variable region comprises the Kabat-defined CDR1, CDR2, andCDR3 sequences of SEQ ID NOs: 5-7, and wherein the light chain variableregion comprises the Kabat-defined CDR1, CDR2, and CDR3 sequences of SEQID NOs: 8-10; b. placing the isolated antibody in a stabilizingformulation to form the stable, aqueous antibody formulation, whereinthe resulting stable, aqueous antibody formulation comprises: i. about 2mg/mL to about 100 mg/mL of the antibody, and ii. about 0.002% to about0.01% polysorbate-20.
 46. An antibody formulation comprising an antibodywherein the antibody comprises a heavy chain variable region and a lightchain variable region, wherein the heavy chain variable region comprisesthe Kabat-defined CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 5-7, andwherein the light chain variable region comprises the Kabat-definedCDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 8-10, wherein the antibodyformulation is essentially free of particles.
 47. An antibodyformulation comprising an antibody wherein the antibody comprises aheavy chain variable region and a light chain variable region, whereinthe heavy chain variable region comprises the Kabat-defined CDR1, CDR2,and CDR3 sequences of SEQ ID NOs: 5-7, and wherein the light chainvariable region comprises the Kabat-defined CDR1, CDR2, and CDR3sequences of SEQ ID NOs: 8-10, wherein the antibody formulation isessentially free of active glutathione S-transferase (GST).
 48. A methodof purifying an antibody wherein the antibody comprises a heavy chainvariable region and a light chain variable region, wherein the heavychain variable region comprises the Kabat-defined CDR1, CDR2, and CDR3sequences of SEQ ID NOs: 5-7, and wherein the light chain variableregion comprises the Kabat-defined CDR1, CDR2, and CDR3 sequences of SEQID NOs: 8-10, the method comprising: a. obtaining a cell culturecomprising the antibody, b. performing affinity chromatography on theantibody, c. performing cation exchange on the antibody, d. performingmixed mode chromatography on the antibody.
 49. A method of treating apulmonary disease or disorder in a subject, the method comprisingadministering a therapeutically effective amount of the antibodyformulation of claim
 24. 50. The method of claim 49, wherein thepulmonary disease or disorder is asthma, COPD, eosinophilic asthma,combined eosinophilic and neutrophilic asthma, aspirin sensitive asthma,allergic bronchopulmonary aspergillosis, acute and chronic eosinophilicbronchitis, acute and chronic eosinophilic pneumonia, Churg-Strausssyndrome, hypereosinophilic syndrome, drug, irritant andradiation-induced pulmonary eosinophilia, infection-induced pulmonaryeosinophilia (fungi, tuberculosis, parasites), autoimmune-relatedpulmonary eosinophilia, eosinophilic esophagitis, Crohn's disease, orcombination thereof.